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Validation of transrectal ultrasonography for assessment of corpora lutea characteristics in pregnant sows and its relationship with litter characteristics at birth

Validation of transrectal ultrasonography for assessment of corpora lutea characteristics in... Validation of transrectal ultrasonography for assessment of corpora lutea charac- teristics in pregnant sows and its relationship with litter characteristics at birth C. L. A. Da Silva,* B. F. A. laurenssen,* e. F. Knol,† B. Kemp,* n. M. Soede* *Adaptation Physiology Group, Wageningen University and Research, Wageningen, The Netherlands, PO Box 338; and †Topigs Norsvin Research Center B.V., Beuningen, The Netherlands, PO Box 86 ABStrAct : In experiment 1 we investigated the after dissection and the average CL diameter based on accuracy of transrectal ultrasonography (TUS) to assess TUS (β = 1.0 ± 0.1 mm/mm, P < 0.0001). This shows the number (OR) and diameter of corpora lutea (CL) in that TUS is not a valid method to assess OR in pregnant 45 and 25 sows, respectively, at 23.4 ± 2.9 d of pregnan- sows but it is a valid method to assess average CL diam- cy. The diameter was calculated as the average diameter eter. In experiment 2, we investigated the relationship of 10 biggest CL. Sows were subsequently slaughtered between the average CL diameter assessed by TUS (n = and OR was assessed by dissection of CL from both 100) at 23.8 ± 2.4 d of pregnancy and average piglet birth ovaries (n = 45) and average diameter of the 10 biggest weight (BW) and observed an increase of 37.6 ± 17.8 g CL was also calculated after measurement of CL with in piglet BW per mm increase in average CL diameter the caliper rule (n = 25). There was a weak relation- measured by TUS (P = 0.04). This relationship is prob- ship between OR counted after dissection of the ovaries ably because larger CL develop from bigger follicles at and OR counted with TUS (β = 0.28 ± 0.01 CL/CL, ovulation, which might have ovulated oocytes of higher P = 0.01), but there was a strong relationship between quality that developed into embryos with higher growth the average CL diameter measured with the caliper rule potential and thus higher birth weight. Key words: corpora lutea, piglet birth weight, pregnancy, sows, transrectal ultrasonography © 2017 American Society of Animal Science. This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Transl. Anim. Sci. 2017.1:507–517 doi:10.2527/tas2017.0057 IntroductIon most assessments of OR in pregnant sows to study its relationship with embryonic characteristics have been Transrectal ultrasonography (TUS) is an established based on post-mortem findings (Vonnahme et al., 2002; technique for assessing ovulation rate (OR) by count- Town et al., 2005; Da Silva et al., 2016; Da Silva et al., ing the number of pre-ovulatory follicles in sows during 2017). It was observed that an increase in OR is related oestrus (Soede et al., 1992; Soede et al., 1998; Lucy et with vital embryos at 35 d of pregnancy with lower pla- al., 2001; Hazeleger et al., 2005; Madej et al., 2005), cental length in sows (Da Silva et al., 2016) and with a and it was used by Gonzalez-Añover et al. (2009) to as- higher variation in weight in gilts (Da Silva et al., 2017). sess OR by counting the number of corpora lutea (CL) This might decrease fetal survival and consequently lit- in Iberian sows at 9 to 11 d after estrus with an accuracy ter size, but might also lead to a decrease in piglet birth of 86.7%. However, TUS has not yet been used to OR weight and birth weight uniformity. Additionally, aver- in Western commercial sows during pregnancy. So far, age CL weight decreased with the increase in OR in gilts (Da Silva et al., 2017), indicating a compromised follicular growth, with oocytes and possibly embryos The authors would like to acknowledge Capes-Brazil, for the of lower quality (Ding and Foxcroft, 1994), compro- first author’s scholarship, Topigs Norsvin for funding the experi- mising embryonic and fetal survival and development. ments and Louisa J. Zak for her comments. Thus, we hypothesized that the number and size of CL Corresponding author: carolina.lima@wur.nl in pregnant sows might be related with litter character- Received August 26, 2017. istics at birth, and the objectives of this study were first Accepted September 8, 2017. Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 508 Da Silva et al. to investigate the accuracy of TUS to assess the number using an Aquila MyVet30 LAB with a convex trans- and average diameter of CL in modern crossbred sows in ducer at 7.5 MHz (Pie Medical/Esaote, Maastricht, The early pregnancy, and second to investigate the relation- Netherlands). Sows were scanned in early pregnancy ship between the CL characteristics evaluated by TUS (21.7 ± 1.1 d of pregnancy in experiment 1, and 23.8 ± and subsequent litter characteristics at birth. 2.4 d in experiment 2). To perform the ultrasonography, sows were placed in individual crates. The scanning MAterIAl And MetHodS procedure involved manual cleaning of the rectum and rectal insertion of a transducer covered with a dispos- The experiments and all measurements were able glove containing scanning gel to prevent the pres- approved by the Animal Welfare Committee of ence of air bubbles in contact with the probe. During Wageningen University and Research Centre in compli- the entire procedure lubricated disposable transrectal ance with the Dutch Law on Animal Experimentation. examination gloves were used, to minimize animal ) was considered as Experiment 1 was conducted at Schothorst Feed discomfort. Ovulation rate (OR TUS Research B.V. (Lelystad, The Netherlands), and at the total number of CL counted on both ovaries with a commercial farm (Nijmegen, The Netherlands). transrectal ultrasonography. In experiment 1, the CL Experiment 2 was conducted at the Pig Innovation counting was performed by 2 examiners separately in a Centre, Sterksel (VIC, Sterksel, The Netherlands). subset of sows (E1 and E2, n = 29), to check for inter- examiner agreement in assessment of OR TUS Also, a movie clip of the examination of each ovary Animals and Housing was saved (25 sows in experiment 1 and 100 sows in Experiment 1. The study included a total of 45 experiment 2) and the diameter of the 5 biggest CL on pregnant multiparous (parity 7.3 ± 3.2, ranging from 2 each ovary (10 per sow) was later assessed, and the aver- to 13) crossbred sows (Yorkshire × Landrace; Topigs age CL diameter (mm) measured by TUS was calculated ). An example of ultrasound images of the Norsvin, Vught, The Netherlands), at 2 different farms (DIAM TUS (farm 1, n = 20 and farm 2, n = 25) in 3 batches each. ovaries can be seen in Fig. 1, and a movie of ovarian ex- Experiment 2. The study included a total of 100 amination has been provided as supplementary material. pregnant multiparous (parity 5.0 ± 1.9, ranging from 2 to 9) crossbred sows (Yorkshire × Landrace; Topigs Dissection of the Ovaries: Ex Vivo Norsvin, Vught, The Netherlands) at 1 farm, which Examination of the Ovaries were used in 6 batches. At the first day after weaning, that took place at d 27.0 ± 3.8 (mean ± SD) of lactation, Sows from experiment 1 (n = 45) were slaugh- the sows were group housed with individual feeding tered at a local abattoir at 29.8 ± 1.9 d of pregnancy stalls, where they received a commercial lactation diet and the uterus and ovaries were collected. Ovulation (NE = 9.50 MJ/kg; CP = 149 g/kg and ileal digestible rate was assessed by dissection of each individual cor- ). lysine = 7.6 g/kg) ad libitum. From the second day post pus luteum present on left and right ovaries (OR DIS weaning to the day of first insemination, sows were In the 25 sows in which CL diameter was measured housed in individual crates, and received 4.5 kg of the with TUS (farm 2), each individual corpus luteum was lactation diet per day. The light schedule consisted of cleaned of connective tissue, their individual diameter 16 consecutive hours of light (100 lux) and 8 h of dark- was measured using a caliper rule and the average and SD ness. After insemination, sows were moved to a gesta- of CL diameter were assessed. Further, each individual tion group housing system in groups of 11 to 45 sows, CL was weighed and the average and SD of CL weight where they received a commercial diet (NE = 9.06 MJ; per sow was calculated. Total luteal mass was calculat- CP = 119 g/kg, ileal digestible lysine = 4.6 g/kg); at ed as the sum of all corpora lutea weights. The diameter 2.8 kg/d from 1 to 34 d of pregnancy, 2.7 kg/d from 35 (mm) of the 5 biggest CL in each ovary (10 per sow) was to 76 d, and 3.3 kg/d from 76 d to farrowing. The light used to estimate the average CL diameter measured after ), and the weight of the 5 CL with schedule during gestation consisted of 12 consecutive dissection (DIAM DIS hours of light (100 lux) and 12 h of darkness. Sows had the highest diameter in each ovary (10 per sow) was used ). free access to water at all times. to assess average CL weight (g) after dissection (WT DIS All CL measurements were done by the same person. Transrectal Ultrasonography Litter Characteristics A list of the abbreviations is provided in Appendix Sows from experiment 2 (n = 100) farrowed and the 1. In experiments 1 and 2 transrectal real time B-mode ultrasonography (TUS) of the ovaries was performed length of gestation, number of piglets born alive (live Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 509 Figure 1. Example of ultrasound image of the ovaries (a and b, left and right ovary respectively), with the white arrows indicating individual corpus luteum. Panels c and d show the left ovary, and e and f show the right ovary, before dissection. Panels g and h show the individual corpus luteum dissected from left and right ovary, respectively. Statistical Analyses born), number of piglets born dead (stillborn), and num- ber of mummified piglets were assessed. Total number All analyses were performed using PROC MIXED of piglets born (litter size) was defined as the sum of in SAS version 9.3 (SAS Inst. Inc., Cary, NC). the number of piglets born alive and dead. Piglets were Experiment 1. Preliminary analyses showed that weighed within 24 h after birth and from this, average there was a farm difference in average OR assessed af- for farm piglet birth weight (total born and live born), within lit- ter dissection [least square means of OR DIS ter SD of piglet birth weight (total born and live born), 1 was 27.5 ± 1.0 and for farm 2 was 21.4 ± 0.9, P < and total litter birth weight were calculated. 0.0001]. Thus, all statistical models for accuracy of CL Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 510 Da Silva et al. counting included the fixed class effect of farm, and the Experiment 2. To assess the effect of sow parity random class effect of batch to account for possible en- on CL, gestation and litter characteristics, parity was vironmental variation. Statistical models for accuracy divided into 3 categories: class 1 (parities 2 and 3, n = of CL diameter measurements did not have farm in the 27), class 2 (parities 4 and 5, n = 35), and class 3 (pari- model since the measurements were done in for a sub- ties 6 to 9, n = 38) and the fixed class effect of parity set of sows coming from the same farm (n = 25). There (parity class 1, 2, and 3) was included in the model was no relationship between the parity of the sows and together with the random effect of batch. the CL measurements done by TUS and after ovarian To assess relationships between the measurements dissection (P > 0.05), so parity was not included in the of CL done by TUS and sow and litter characteristics , to- models. In all models, fixed class effects were corrected at birth, the continuous fixed effect of DIAM TUS with Bonferroni and if nonsignificant were removed gether with the fixed class effect of parity (1, 2, and 3) from the models. Interactions were never significant and the fixed class effect of litter size [class 1 (9 to 16 and were therefore removed from the models. piglets born, n = 34); class 2 (17 to 19 piglets born, n as as- First, the relationship between the OR = 36); and class 3 (20 to 26 piglets born, n = 30)] and TUS sessed by the 2 examiners separately (E1 and E2) was their interaction, were assessed on litter characteristics. investigated to check for inter-examiner agreement. In all models, batch was included as a random class ef- count- For this, the continuous linear effect of OR fect to account for possible environmental variation. If TUS counted by E1. ed by E2 was assessed on OR nonsignificant, the interactions and the fixed class ef- TUS Analyses on the accuracy of TUS were done using fects were removed from the models. Residuals from the data of E1 (n = 45). First, the difference between all models approximated normality based on skewness and OR in number of CL counted was cal- OR and kurtosis. Results are presented as the regression co- TUS DIS culated. Further, to check the accuracy of TUS in as- efficients (β) with their SE for continuous fixed effects relates with OR ) sessing OR (i.e., how does OR and as least squares means and their SE for fixed class TUS DIS was assessed on the continuous linear effect of OR effects. Results are considered significant at P ≤ 0.05. DIS . To check if the accuracy of TUS was affected OR TUS the continuous fixed effect of OR was by OR reSultS DIS DIS and OR . assessed on the difference between OR TUS DIS Regarding CL diameter measurements, the differ - Experiment 1 ence between average CL diameter estimated based on TUS and after ovarian dissection were assessed (i.e., The averages and SD of the CL, litter and sows – DIAM ). Further, to check the accuracy DIAM characteristics are presented in Table 1. In experiment TUS DIS of TUS in measuring the diameter of the 10 biggest 1, the total number of CL (mean ± SD) assessed by ) was 23.4 ± 5.8 (ranging from 13 to 34) CL, the continuous fixed effect of the average diam - TUS (OR TUS ) was eter of the 10 biggest CL at dissection (DIAM and the number counted after dissection of the ovaries DIS ) was 24.1 ± 5.3 (ranging from 13 to 37). The assessed on the average diameter of the 10 biggest CL (OR DIS ). To check if the accuracy of TUS by TUS (DIAM average diameter of the 10 biggest CL measured with TUS , the ) was 10.3 ± 0.7 mm and after dis- in measuring CL diameter was affected by OR TUS (DIAM DIS TUS was assessed on the ) was 10.3 ± 0.6 mm. The average continuous fixed effect of OR section (DIAM DIS DIS and DIAM . difference between DIAM diameter of all CL dissected was 9.8 ± 0.7 mm with an TUS DIS Also, to investigate the relationship between OR SD of 0.8 ± 0.4 mm. The average weight of the 10 big- ) was 0.40 ± 0.07 g, and the average and CL size, the continuous linear and quadratic effect gest CL (WT DIS was assessed on DIAM , DIAM and of OR weight of all CL dissected was 0.38 ± 0.07g. Average DIS TUS DIS ). on average CL weight after dissection (WT total luteal mass per sow was 7.9 ± 1.5g. DIS Moreover, aiming to check the relationship be- tween OR and characteristics of all CL dissected, the Relationship between CL Characteristics was continuous linear and quadratic effect of OR after Ovarian Dissection and with TUS DIS assessed on the average and standard deviation of CL diameter and weight of all CL, and on total luteal mass. Results show that there was a strong relationship assessed by the 2 examiners, E2 and Residuals of all models approximated normality between OR TUS based on skewness and kurtosis. Results are presented as E1 (β = 0.87 ± 0.09 CL E1/CL E2, P < 0.0001, R the regression coefficients (β) with their standard errors 0.93, Fig. 2A), showing that there was inter-observer (SE) for the continuous linear and quadratic fixed effects agreement in the assessment of OR using TUS. and as least square means and their SE for fixed class ef- Differences in CL number measured with TUS and fects. Results are considered significant at P ≤ 0.05. after ovarian dissection are presented in Fig. 3. The av- Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 511 Figure 2. Panel A, predicted relationship between OR assessed by TUS performed by examiner 2 and the OR assessed by TUS performed by exam- iner 1 [(n = 29), β = 0.87 ± 0.09 CLTUS E1/CLTUS E2, P < 0.0001, farm P = 0.12]. Panel B, predicted relationship between the OR assessed by E1 by TUS and OR after dissection [β = 0.28 ± 0.01 TUS/DIS, P = 0.01, farm P = 0.0002, (n = 45)]. Panel C; predicted relationship between the average diameter of the 10 biggest CL assessed after ovarian dissection and the average diameter of the 10 biggest CL assessed by TUS [(n = 25), β = 1.0 ± 0.07 mm TUS/ mm dissected, P < 0.0001]. Panel D, predicted relationship between the OR after dissection and the average CL diameter [TUS = 0.49 ± 0.19 (P = 0.02) – 0.01 ± 0.005 (P = 0.01) and DISS = 0.47 ± 0.18 (P = 0.02) – 0.01 ± 0.004 (P = 0.01)]. Statistical models included the fixed class effect of farm (n = 2, for panels a and b), and the random effect of batch (n = 6). Data points (♦) represent the predicted values estimated as the difference with the model residuals. sows the difference was 6 or more. So, there was not a close relationship between OR and OR [β = 0.28 DIS TUS ± 0.01 TUS/DISS, P = 0.01, farm 1 = 28.0 ± 1.3 and = 0.79; Fig. 2B]. farm 2 = 20.5 ± 1.2; P = 0.0002, R Furthermore, there was a positive linear relationship and the difference in OR assessed by between OR DIS TUS and after dissection (β = 0.72 ± 0.10 difference/CL = 0.68). So, dissected, P < 0.0001, farm P = 0.0002, R TUS is not an accurate method to estimate OR in sows at early pregnancy, and there is an increase in the inac- curacy of TUS with an increase in OR. Differences in the average CL diameter measured by TUS and the average CL diameter measured after – DIAM ) were as- ovarian dissection (DIAM Figure 3. Summary of the differences between the number of corpora TUS DIS lutea (CL) counted with transrectal ultrasonography (OR ) and the num- sessed. The average ± SD of the difference between the TUS ber of CL counted after slaughter and dissection of the ovaries (OR ) in DIS and DIAM was 0.02 ± 0.20 mm (rang- DIAM TUS DIS 45 multiparous sows at early pregnancy. Underestimations are shown in ing from –0.36 up to 0.34 mm difference). There was light gray and overestimations are shown in dark gray. a positive linear relationship between the DIAM DIS (β = 1.00 ± 0.07 mm TUS/mm DIS; and DIAM TUS erage ± SD of the difference in OR assessed by TUS = 0.96; Fig. 2C). Moreover, there was P < 0.0001, R and the difference be- and OR assessed after ovarian dissection was 0.7 ± 4.7 no relationship between OR DIS and DIAM (β = 0.005 ± 0.01 mm CL (ranging from –8 up to +12 CL difference). Results tween DIAM TUS DIS differed from = 0.44; P = 0.58). So, TUS show that in 24.4% of the sows OR difference/CL dissected, R TUS in by 0 or 1 CL; in 28.9% of the sows the dif- OR is an accurate method to estimate the average diameter DIS ference was 2 or 3, in 24.4% 4 or 5 and in 22.2% of the of the 10 biggest CL in sows in early pregnancy and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 512 Da Silva et al. the accuracy of the measurements done by TUS is not OR (P = 0.03)], which shows a maximum average DIS related with an increase in OR. CL diameter of 10.1 mm at 18 ovulations. There was and the and total Regarding the relationship between OR a negative linear relationship between OR DIS DIS average CL diameter and weight of the 10 biggest CL, average CL weight (β = - 0.01 ± 0.03 g/CL dissected, P and was not related with the SD there was a quadratic relationship between OR = 0.002). However, OR DIS DIS [0.49 ± 0.2 × OR (P = 0.02)– 0.01 ± the DIAM in CL diameter (P = 0.85) and with the SD in CL weight TUS DIS 2 2 (P = 0.01), R = 0.76; Fig. 2D] and 0.0004 × OR (P = 0.12). Furthermore, there was a positive linear re- DIS [0.47 ± 0.2 × OR (P = 0.02) – 0.01 ± 0.0004 and total luteal mass (β = + DIAM lationship between OR DIS DIS DIS 2 2 (P = 0.01), R = 0.69; Fig. 2D], which shows × OR 0.19 ± 0.1 g/CL dissected, P = 0.003). So, the total aver- DIS a maximum average diameter of the 10 biggest CL of age diameter and weight of the CL is lower in sows with 10.5 mm at 19 ovulations for measurements done with a higher OR, but there is no increase in variation in CL and DIAM , TUS and also after dissection (DIAM size with the increase in OR. Also, the total luteal mass TUS DIS respectively). Also, there was a negative linear relation- increases linearly with the increase in OR, despite the and the average weight of the 10 ship between OR decrease in average CL weight. DIS = 0.51; biggest CL (β = - 0.01 ± 0.003 g/CL dissected, R P = 0.01). So, the average diameter and weight of the 10 Experiment 2 biggest CL is lower in sows with a higher OR. There was a quadratic relationship between OR The averages and SD of sows, CL and litter char- DIS and the average diameter of all CL measured after dis- acteristics are presented in Table 1. The average CL (P = 0.05)– 0.01 ± 0.01 × section [0.43 ± 0.2 × OR diameter measured with TUS was 8.4 ± 0.8 mm, rang- DIS t able 1. Summary statistics of sows, corpus luteum and litter characteristics Variables n Mean SD Min Max Experiment 1 Parity 45 7.3 3.2 2 13 TUS pregnancy age, d 45 23.4 2.9 20 28 Slaughter pregnancy age, d 45 29.8 1.9 27 32 Ovulation rate by TUS 45 23.4 5.8 13 34 Ovulation rate after dissection 45 24.1 5.3 13 37 Average CL diameter TUS , mm 25 10.3 0.73 8.4 11.7 Average CL diameter DISS , mm 25 10.3 0.64 8.7 11.5 Average CL weight DISS , g 25 0.40 0.07 0.22 0.56 Total average CL diameter , mm 25 9.8 0.7 7.8 11.0 Total average CL weight , g 25 0.38 0.1 0.20 0.51 Total luteal mass , g 25 7.9 1.5 5.2 10.7 Experiment 2 Parity 100 5.0 1.9 2 9 TUS pregnancy age, d 100 23.8 2.4 21 29 Gestational length, d 100 115 1.7 111 120 Average CL diameter TUS , mm 100 8.4 0.8 5.5 10.5 Litter size 100 17.9 3.0 9 26 Number of live born 100 16.3 2.9 6 23 Number of stillborn 100 1.53 1.8 0 9 Number of mummies 100 0.42 0.8 0 5 Average piglet BW , g 100 1277 165 914 1618 SD BW , g 100 303 76 161 555 Average live born piglets BW , g 100 1299 167 933 1674 SD BW live born , g 100 292 76 115 492 Litter BW, kg 100 23 4.0 15 30 Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured by Transrectal ultrasonography (TUS). Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured with caliper rule after slaughter of the sows and ovarian dissection. Average weight of the 5 corpora lutea in each ovary (10 per sow) that had the highest diameter measured after ovarian dissection. Average diameter and weight calculated based on the measurement of all corpora lutea dissected from each ovary. Sum of the weight of all the corpora lutea dissected from the ovaries. Litter size is the sum of piglets born alive (live born) and stillborn piglets. Piglets were weighed within 24 h after birth. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 513 ing from 5.5 to 10.5 mm. The average total number of piglets born was 17.9 ± 3.0, and 16.3 ± 2.9 were born alive. The average birth weight (BW) of the total piglets born was 1,277 ± 165 g and the average within litter BW variation was 303 ± 76 g, the average BW of the piglets born alive was 1,299 ± 167 g and average within litter BW variation was 292 ± 76 g. Effect of Parity on Gestation Length, CL and Litter Characteristics Parity did not affect the gestation length, the aver - age diameter of the 10 biggest CL measured by TUS ), litter size, average and SD piglet birth (DIAM TUS weight or litter weight (P > 0.05). Sows in parity 6 up to 9 had a higher (2.13 ± 0.3, P = 0.02) number of stillborn piglets than sows in parity 2 up to 3 (0.86 ± 0.4) and 4 up to 5 (1.34 ± 0.3). Relationship between CL Diameter and Litter Characteristics and litter charac- Relationships between DIAM TUS teristics are presented in Table 2. There was a positive and average pig- linear relationship between DIAM TUS let BW [(β = + 37.6 ± 17.8 g/mm; P = 0.04, Fig. 4A) Figure 4. Panel A: estimated least square means for the effect of average + c value dependent on litter size (P < 0.0001): 9 to 16 CL diameter classes [5.5 to 7.8 mm (n = 23); 7.9 to 8.9 mm (n = 47); and 9.0 to 10.5 mm (n = 30)] on BW of total piglets born [P = 0.04; litter size class P piglets = 1367 ± 25 g, 17 to 19 piglets = 1,280 ± 24 g, < 0.0001]. Panel B: estimated least square means for the effect of CL diameter and 20 to 26 piglets = 1,171 ± 26 g]. There was also a classes on standard deviation (SD) of BW of the total piglets born (P = 0.02). and the positive linear relationship between DIAM TUS Statistical models included the fixed class effect of litter size [LS, 8 to 18 pig- SD in BW of the total born piglets (β = 24.3 ± 10.4 g/ lets born (n = 35); 17 to 19 piglets born (n = 36) and 20 to 26 piglets born (n = 30)] and its interactions, which were excluded from the models when not mm; P = 0.02, Fig. 4B) and the SD in BW of the piglets significant (P > 0.05). Significant differences between classes are indicated by born alive (β = 27.3 ± 10.5 g/mm, P = 0.01). There were letters above the columns and the error bars indicate the SE of the estimates. t able 2. Relationship between average corpora lutea diameter measured by transrectal ultrasonography (DIAM ) and litter characteristics at birth TUS DIAM , mm Litter size Parity TUS Dependent variables β (SEM) P-value P-value P-value Gestational length, d 0.48 (0.26) 0.06 ns ns Litter size –0.098 (0.43) 0.82 – ns Number of live born –0.084 (0.43) 0.84 – ns Number of stillborn –0.015 (0.22) 0.95 0.02 0.01 Number of mummies –0.013 (0.10) 0.90 ns ns Average piglet BW , g 37.57 (17.84) 0.04 < 0.0001 ns SD BW , g 24.25 (10.37) 0.02 ns ns Average live born piglets BW , g 32.98 (18.41) 0.08 < 0.0001 ns SD BW live born , g 27.33 (10.52) 0.01 ns ns Litter BW, kg 0.81 (0.47) 0.09 < 0.0001 ns Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured by TUS. ns = P > 0.05. Litter size is the sum of piglets born alive (live born) and stillborn piglets. Interaction between parity (PC) and litter size (LS) classes (P = 0.002): PC1 × LS1 (n = 9): 0.67 ± 0.55b; PC2 × LS1 (n = 12): 0.48 ± 0.48b; PC3 × LS1 (n = 6): 1.54 ± 0.47ab; PC2 × LS2 (n = 12): 1.02 ± 0.49b; PC2 × LS2 (n = 12): 2.01 ± 0.49b; PC3 × LS2 (n = 11): 0.92 ± 0.49ab; PC1 × LS3 (n = 13): 0.81 ± 0.66b; PC2 × LS3 (n = 12): 1.58 ± 0.51ab; PC3 × LS3 (n = 13): 3.83 ± 0.47a. Piglets were weighed within 24 h after birth (BW). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 514 Da Silva et al. no interactions between DIAM , litter size classes stroma, which makes visualization of each individual CL TUS and parity classes. So, an increase in the average CL more difficult. Moreover, despite the fact that transrectal diameter in early pregnancy is related with an increase ultrasonography is believed to provide clearer images of in average piglet birth weight and in within litter birth the ovaries due to less interference of intestinal tissues weight variation. (Kauffold and Althouse, 2007), is still possible that the ovaries were only partially visible due to interference of dIScuSSIon the intestinal and uterine tissue of the pregnant sows. Overestimations, on the other hand might be ex- This is, to our knowledge, the first study that in- plained by counting CL from part of an ovary 2 times, vestigated the accuracy of transrectal ultrasonography which might happen due to the proximity of the 2 ova- to assess CL number and diameter in sows in early ries during the examination and the difficulty in distin- pregnancy and investigated the relationship between guishing between the 2 separate ovaries. The lack of CL diameter measured by transrectal ultrasonography accuracy of transrectal ultrasonography in assessing the and litter characteristics at birth. number of CL in pregnant sows might also be related Transrectal ultrasonography did not provide an ac- with the high number of CL present in each ovary (high curate estimation of the number of corpora lutea (OR) ovulation rate), where crowding of the ovaries makes in sows in early pregnancy. The difference between the it more difficult to differentiate the CL. Gonzalez- OR assessed after ovarian dissection and with TUS was Añover et al. (2009) investigated the accuracy of tran- on average 0.7 ± 4.7 CL, ranging from an underestima- srectal ultrasonography in accessing the number of CL tion of 8 CL up to an overestimation of 12 CL. Moreover, in Iberian sows in the mid luteal phase (average ovu- only in 24.4% of the sows, the difference between OR lation rate of 6.0 ± 1.3), and achieved accuracy close assessed with TUS and after ovarian dissection was of to 100% in ovaries with 5 CL or less, which decreased only 1 CL, with 46.7% of the TUS estimations differing with the increase in number of CL present per ovary. with more than 4 CL. This inaccuracy occurred almost A decrease in accuracy with the increase in OR was equally due to under and over estimations of the number also observed by Soede et al. (1992) and Bolarin et al. of CL assessed after dissection of the ovaries (33.3 and (2009) when counting the number of pre-ovulatory fol- 42.2%, respectively). Underestimations are probably re- licles. In the present study (average OR of 24.1 ± 5.3) lated with the difficulty in visualizing all individual CL. there was also a decrease in accuracy of TUS (differ - Ultrasound machines uses high frequency sound waves ence between OR counted by TUS and after slaughter) and their echo to produce images (Pierson et al., 1988) with the increase in OR after ovarian dissection. This and different tissues have different abilities to reflect the could be related with the decrease in CL size with the sound waves (Pierson et al., 1988). Thus, characteristics increase in ovulation rate. Average CL diameter in sows of a tissue determine the proportion of the sound wave with 13 up to 22 ovulations was predicted to be 10.3 that will be reflected, which will then be represented on mm, decreasing to 8.7 mm in sows with more than 23 the ultrasound image display by dots of different shades ovulations. The same is true for the average CL weight, of gray, varying from black to white. Liquids, like fol- which can be 0.13g smaller in sows with more than 23 licular fluid, do not reflect sound waves (non-echogenic) ovulations in comparison with sows with 13 up to 22 and therefore the image appears as black on the screen, ovulations. A decrease in average CL weight (and thus in contrast with the surrounding ovarian tissue, that re- size, this study) with the increase in ovulation rate was flects part of the sound waves (echogenic) and can be also observed in gilts at 35 d of pregnancy (Da Silva seen as different shades of gray (Pierson et al., 1988). et al., 2017). This indicates that with a higher OR, not This contrast facilitates visualization of individual fol- only the ovaries are more crowded, but also individu- licles and explains the high accuracy of transrectal ul- al CL are smaller, which might increase the difficulty trasonography in assessing the number of pre-ovulatory in visualization of the individual CL, thus decreasing follicles in sows. Soede et al. (1992) described a differ - the accuracy of counting CL with TUS. The reason for ence of only 0.4 ± 1.8 between the number of follicles the decrease in CL size with the increase in ovulation counted by transrectal ultrasonography and the number rate is not known. During follicular growth, recruited of CL counted after slaughter of the sows (18.6 ± 3.5). follicles respond to FSH by increasing the production Also, Bolarin et al. (2009) observed a significant correla- of estradiol-17β (Britt and Findlay, 2002; Drummond, tion between the number of pre-ovulatory follicles (6 to 2006). The increase in estradiol-17β (E2) produc- 10 mm) counted per ovary with transrectal ultrasonogra- tion leads to an increase in granulosa cell number and phy and with laparoscopy (r = 0.98, P < 0.001). Corpora therefore to further follicular development (Drummond lutea, however, are tissue filled glands and are echogenic, and Findlay, 1999). So, together with the increase in surrounded by the also echogenic tissue of the ovarian E2 production, follicles increase in size. E2 production Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 515 increases until it reaches a certain systemic threshold elongation is established by the conceptus (Geisert et al., concentration that triggers a pre-ovulatory GnRH/LH 2014) and more advanced embryos [derived from the surge that subsequently triggers ovulation (Drummond more developed oocytes, Pope et al. (1990)] elongate and Findlay, 1999; Drummond, 2006). With more fol- earlier and will have an increased uterine implantation licles recruited from the pool (i.e., higher OR), and length (Geisert et al., 1982a). The uterine implantation therefore more follicles producing E2, the threshold of length is related with the placental length of vital em- E2 necessary to trigger ovulation might occur when fol- bryos at 35 d of pregnancy in sows [β = 0.98 ± 0.14 cm/ licles are of smaller size than in sows with a lower OR. cm, P < 0.0001. C. L. A. Da Silva, unpublished results], Indeed, Knox et al. (2003) observed that systemic E2 which will possibly favor further fetal development and levels were the same at days –1 to +1 relative to the LH consequently piglet birth weight. peak (d 0) in gilts selected for high OR (OR 18.8 ± 0.4, Corpora lutea produce progesterone, which is of E2 37.9 ± 3.4 ng/mL) and in gilts of the control line (OR primary importance for maintenance of the pregnancy 14.3 ± 0.6, E2 44.7 ± 3.6 ng/mL), indicating that the in the pig (Spencer et al., 2004). So, it could be hypoth- threshold of E2 that precedes ovulation is independent esized that higher CL size in pregnant sows is related of OR. Soede et al. (1998) observed that the average with higher progesterone production favoring embry- volume of pre-ovulatory follicles at ovulation per sow onic growth and piglet birth weight. However, systemic was significantly correlated with the average CL weight progesterone levels in 238 gilts at 35 d of pregnancy (C. at 5 d of pregnancy (r = 0.28, P < 0.01), and Wientjes et L. A Da Silva, unpublished results), were not related al. (2012) observed that each mm increase in follicle di- with average CL weight (P = 0.69). It could also be pos- ameter at ovulation was related with 1.23 mm increase sible that average CL weight and piglet birth weight in CL diameter (P = 0.03) in multiparous sows at 10 d have a common origin in early pregnancy. Corpora lu- of pregnancy. Thus, high OR sows may have smaller tea regression occurs on d 15 to 16 of the oestrus cycle follicles at ovulation that develop into smaller CL. due to the increase in pulsatile endometrial secretion of We also investigated the validity of TUS to measure prostaglandin F2-ɑ (Moeljono et al., 1976). Thus, preg- CL diameter in sows. Results show that TUS is an ac- nancy recognition requires the development and main- curate method to assess CL diameter in sows at 3 to 4 wk tenance of CL beyond the luteal phase, and is a result of of pregnancy. There was a strong relationship between oestrogens (mainly estradiol-17β, E2) secretion by the the CL diameter measured by transrectal ultrasonogra- conceptuses on d 11 and 12, followed by a second peak phy and the CL diameter measured after dissection of between d 15 and 25 through 30 of pregnancy (Geisert the ovaries. Further, we investigated the relationship be- et al., 1990). Conceptuses E2 increases luteal LH re- tween average CL diameter assessed by transrectal ultra- ceptor concentration, and together with Il-β1, favors the sonography in early pregnancy and litter characteristics production of luteoprotective PGE2, which stimulates at birth and observed that there is an increase in average the expression of Vascular Endothelial Growth Factor piglet birth weight with an increase in average diameter (VEGF) in luteal cells, increasing luteal permeability of the 10 biggest CL. This might indicate that sows with and delivery of cholesterol to the luteal cells (Ziecik et a higher average CL diameters ovulated oocytes of better al., 2011), which might favor CL growth. So, concep- quality, that developed into embryos with higher growth tus development at the time of elongation and maternal potential and consequently into piglets with higher birth recognition of pregnancy may influence luteal vascu- weight. Indeed, in a recent study with 390 gilts slaugh- larization (through the effects of PGE2 and VEGF), tered at 35 d of pregnancy we found that there is an in- and thereby increase CL size. However, further inves- crease of 2.3 g in the weight of the vital embryos per tigations are needed to understand the mechanisms un- gram of increase in average CL weight [P = 0.001; C. derlying the relationship between CL size, embryonic L. A. Da Silva, unpublished results]. Heavier embryos growth and piglets birth weight. at d 35 of pregnancy might develop into heavier piglets An increase in average CL diameter was not only at birth. Larger/heavier CL develop from larger follicles related with average piglet birth weight as discussed at ovulation, as discussed above. Larger follicles at ovu- above, but also with an increase in within litter varia- lation are known to release oocytes with superior qual- tion in piglet birth weight. Within litter piglet birth ity due to a more advanced maturational status (Hunter, weight variation increases with the increase in litter size 2000; Gandolfi et al., 2005), which might lead to the (Milligan et al., 2002; Quiniou et al., 2002; Wolf et al., development of embryos with higher growth potential 2008), and is seen as a consequence of uterine crowding (Krisher, 2004). At d 11 and 12 of pregnancy, pig concep- on placental development in the early post implantation tuses transition from spherical to tubular and filamentous period (Foxcroft et al., 2006). However, in the current blastocysts in a process called elongation (Geisert et al., study, we did not observe a higher variation in piglet 1982a; Geisert et al., 1982b). Timing of rapid conceptus birth weight in bigger litters. 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But it is also Interrelationships between preimplantation development of the possible that this increase in variation is caused by a pig blastocyst and uterine endometrial secretions. Biol. Reprod. limitation in uterine capacity imposed by the increase 27:925–939. doi:10.1095/biolreprod27.4.925 competition during fetal growth, i.e., an increase in Geisert, R. D., J. W. Brookbank, R. M. Roberts, and F. W. Bazer. 1982b. Establishment of pregnancy in the pig: II. Cellular remodeling of competition for blood flow and nutrient uptake between the porcine blastocyst during elongation on day 12 of pregnancy. littermates, which might also lead to variation in fetal Biol. Reprod. 27:941–955. doi:10.1095/biolreprod27.4.941 growth and in piglet birth weight (Ford et al., 2002). Geisert, R. D., M. C. Lucy, J. J. Whyte, J. W. Ross, and D. J. In conclusion, transrectal ultrasonography is not a Mathew. 2014. 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Anim. 46:31–41. doi:10.1111/j.1439-0531.2011.01843.x APPendIX 1: lISt oF ABBreVIA tIonS Abbreviations Description TUS Transrectal ultrasonography OR Ovulation rate, i.e. the total number of CL present in both ovaries CL Corpora lutea OR Total number of corpora lutea counted on both ovaries with transrectal ultrasonography TUS E1 Transrectal ultrasonography examiner one E2 Transrectal ultrasonography examiner two DIAM The average diameter of the 10 biggest corpora lutea measured by transrectal ultrasonography in a sow TUS OR Total number of corpora lutea counted after slaughter and dissection of the ovaries for individual corpora lutea DIS DIAM The average diameter of the 10 biggest corpora lutea measured with a caliper rule after slaughter and dissection of the ovaries for DIS individual corpora lutea WT The average weight of the 10 biggest corpora lutea measured after slaughter and dissection of the ovaries for individual corpora lutea DIS LS Litter size, i.e. the sum of the number of piglets born alive and dead BW Piglet birth weight Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Validation of transrectal ultrasonography for assessment of corpora lutea characteristics in pregnant sows and its relationship with litter characteristics at birth

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Abstract

Validation of transrectal ultrasonography for assessment of corpora lutea charac- teristics in pregnant sows and its relationship with litter characteristics at birth C. L. A. Da Silva,* B. F. A. laurenssen,* e. F. Knol,† B. Kemp,* n. M. Soede* *Adaptation Physiology Group, Wageningen University and Research, Wageningen, The Netherlands, PO Box 338; and †Topigs Norsvin Research Center B.V., Beuningen, The Netherlands, PO Box 86 ABStrAct : In experiment 1 we investigated the after dissection and the average CL diameter based on accuracy of transrectal ultrasonography (TUS) to assess TUS (β = 1.0 ± 0.1 mm/mm, P < 0.0001). This shows the number (OR) and diameter of corpora lutea (CL) in that TUS is not a valid method to assess OR in pregnant 45 and 25 sows, respectively, at 23.4 ± 2.9 d of pregnan- sows but it is a valid method to assess average CL diam- cy. The diameter was calculated as the average diameter eter. In experiment 2, we investigated the relationship of 10 biggest CL. Sows were subsequently slaughtered between the average CL diameter assessed by TUS (n = and OR was assessed by dissection of CL from both 100) at 23.8 ± 2.4 d of pregnancy and average piglet birth ovaries (n = 45) and average diameter of the 10 biggest weight (BW) and observed an increase of 37.6 ± 17.8 g CL was also calculated after measurement of CL with in piglet BW per mm increase in average CL diameter the caliper rule (n = 25). There was a weak relation- measured by TUS (P = 0.04). This relationship is prob- ship between OR counted after dissection of the ovaries ably because larger CL develop from bigger follicles at and OR counted with TUS (β = 0.28 ± 0.01 CL/CL, ovulation, which might have ovulated oocytes of higher P = 0.01), but there was a strong relationship between quality that developed into embryos with higher growth the average CL diameter measured with the caliper rule potential and thus higher birth weight. Key words: corpora lutea, piglet birth weight, pregnancy, sows, transrectal ultrasonography © 2017 American Society of Animal Science. This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Transl. Anim. Sci. 2017.1:507–517 doi:10.2527/tas2017.0057 IntroductIon most assessments of OR in pregnant sows to study its relationship with embryonic characteristics have been Transrectal ultrasonography (TUS) is an established based on post-mortem findings (Vonnahme et al., 2002; technique for assessing ovulation rate (OR) by count- Town et al., 2005; Da Silva et al., 2016; Da Silva et al., ing the number of pre-ovulatory follicles in sows during 2017). It was observed that an increase in OR is related oestrus (Soede et al., 1992; Soede et al., 1998; Lucy et with vital embryos at 35 d of pregnancy with lower pla- al., 2001; Hazeleger et al., 2005; Madej et al., 2005), cental length in sows (Da Silva et al., 2016) and with a and it was used by Gonzalez-Añover et al. (2009) to as- higher variation in weight in gilts (Da Silva et al., 2017). sess OR by counting the number of corpora lutea (CL) This might decrease fetal survival and consequently lit- in Iberian sows at 9 to 11 d after estrus with an accuracy ter size, but might also lead to a decrease in piglet birth of 86.7%. However, TUS has not yet been used to OR weight and birth weight uniformity. Additionally, aver- in Western commercial sows during pregnancy. So far, age CL weight decreased with the increase in OR in gilts (Da Silva et al., 2017), indicating a compromised follicular growth, with oocytes and possibly embryos The authors would like to acknowledge Capes-Brazil, for the of lower quality (Ding and Foxcroft, 1994), compro- first author’s scholarship, Topigs Norsvin for funding the experi- mising embryonic and fetal survival and development. ments and Louisa J. Zak for her comments. Thus, we hypothesized that the number and size of CL Corresponding author: carolina.lima@wur.nl in pregnant sows might be related with litter character- Received August 26, 2017. istics at birth, and the objectives of this study were first Accepted September 8, 2017. Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 508 Da Silva et al. to investigate the accuracy of TUS to assess the number using an Aquila MyVet30 LAB with a convex trans- and average diameter of CL in modern crossbred sows in ducer at 7.5 MHz (Pie Medical/Esaote, Maastricht, The early pregnancy, and second to investigate the relation- Netherlands). Sows were scanned in early pregnancy ship between the CL characteristics evaluated by TUS (21.7 ± 1.1 d of pregnancy in experiment 1, and 23.8 ± and subsequent litter characteristics at birth. 2.4 d in experiment 2). To perform the ultrasonography, sows were placed in individual crates. The scanning MAterIAl And MetHodS procedure involved manual cleaning of the rectum and rectal insertion of a transducer covered with a dispos- The experiments and all measurements were able glove containing scanning gel to prevent the pres- approved by the Animal Welfare Committee of ence of air bubbles in contact with the probe. During Wageningen University and Research Centre in compli- the entire procedure lubricated disposable transrectal ance with the Dutch Law on Animal Experimentation. examination gloves were used, to minimize animal ) was considered as Experiment 1 was conducted at Schothorst Feed discomfort. Ovulation rate (OR TUS Research B.V. (Lelystad, The Netherlands), and at the total number of CL counted on both ovaries with a commercial farm (Nijmegen, The Netherlands). transrectal ultrasonography. In experiment 1, the CL Experiment 2 was conducted at the Pig Innovation counting was performed by 2 examiners separately in a Centre, Sterksel (VIC, Sterksel, The Netherlands). subset of sows (E1 and E2, n = 29), to check for inter- examiner agreement in assessment of OR TUS Also, a movie clip of the examination of each ovary Animals and Housing was saved (25 sows in experiment 1 and 100 sows in Experiment 1. The study included a total of 45 experiment 2) and the diameter of the 5 biggest CL on pregnant multiparous (parity 7.3 ± 3.2, ranging from 2 each ovary (10 per sow) was later assessed, and the aver- to 13) crossbred sows (Yorkshire × Landrace; Topigs age CL diameter (mm) measured by TUS was calculated ). An example of ultrasound images of the Norsvin, Vught, The Netherlands), at 2 different farms (DIAM TUS (farm 1, n = 20 and farm 2, n = 25) in 3 batches each. ovaries can be seen in Fig. 1, and a movie of ovarian ex- Experiment 2. The study included a total of 100 amination has been provided as supplementary material. pregnant multiparous (parity 5.0 ± 1.9, ranging from 2 to 9) crossbred sows (Yorkshire × Landrace; Topigs Dissection of the Ovaries: Ex Vivo Norsvin, Vught, The Netherlands) at 1 farm, which Examination of the Ovaries were used in 6 batches. At the first day after weaning, that took place at d 27.0 ± 3.8 (mean ± SD) of lactation, Sows from experiment 1 (n = 45) were slaugh- the sows were group housed with individual feeding tered at a local abattoir at 29.8 ± 1.9 d of pregnancy stalls, where they received a commercial lactation diet and the uterus and ovaries were collected. Ovulation (NE = 9.50 MJ/kg; CP = 149 g/kg and ileal digestible rate was assessed by dissection of each individual cor- ). lysine = 7.6 g/kg) ad libitum. From the second day post pus luteum present on left and right ovaries (OR DIS weaning to the day of first insemination, sows were In the 25 sows in which CL diameter was measured housed in individual crates, and received 4.5 kg of the with TUS (farm 2), each individual corpus luteum was lactation diet per day. The light schedule consisted of cleaned of connective tissue, their individual diameter 16 consecutive hours of light (100 lux) and 8 h of dark- was measured using a caliper rule and the average and SD ness. After insemination, sows were moved to a gesta- of CL diameter were assessed. Further, each individual tion group housing system in groups of 11 to 45 sows, CL was weighed and the average and SD of CL weight where they received a commercial diet (NE = 9.06 MJ; per sow was calculated. Total luteal mass was calculat- CP = 119 g/kg, ileal digestible lysine = 4.6 g/kg); at ed as the sum of all corpora lutea weights. The diameter 2.8 kg/d from 1 to 34 d of pregnancy, 2.7 kg/d from 35 (mm) of the 5 biggest CL in each ovary (10 per sow) was to 76 d, and 3.3 kg/d from 76 d to farrowing. The light used to estimate the average CL diameter measured after ), and the weight of the 5 CL with schedule during gestation consisted of 12 consecutive dissection (DIAM DIS hours of light (100 lux) and 12 h of darkness. Sows had the highest diameter in each ovary (10 per sow) was used ). free access to water at all times. to assess average CL weight (g) after dissection (WT DIS All CL measurements were done by the same person. Transrectal Ultrasonography Litter Characteristics A list of the abbreviations is provided in Appendix Sows from experiment 2 (n = 100) farrowed and the 1. In experiments 1 and 2 transrectal real time B-mode ultrasonography (TUS) of the ovaries was performed length of gestation, number of piglets born alive (live Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 509 Figure 1. Example of ultrasound image of the ovaries (a and b, left and right ovary respectively), with the white arrows indicating individual corpus luteum. Panels c and d show the left ovary, and e and f show the right ovary, before dissection. Panels g and h show the individual corpus luteum dissected from left and right ovary, respectively. Statistical Analyses born), number of piglets born dead (stillborn), and num- ber of mummified piglets were assessed. Total number All analyses were performed using PROC MIXED of piglets born (litter size) was defined as the sum of in SAS version 9.3 (SAS Inst. Inc., Cary, NC). the number of piglets born alive and dead. Piglets were Experiment 1. Preliminary analyses showed that weighed within 24 h after birth and from this, average there was a farm difference in average OR assessed af- for farm piglet birth weight (total born and live born), within lit- ter dissection [least square means of OR DIS ter SD of piglet birth weight (total born and live born), 1 was 27.5 ± 1.0 and for farm 2 was 21.4 ± 0.9, P < and total litter birth weight were calculated. 0.0001]. Thus, all statistical models for accuracy of CL Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 510 Da Silva et al. counting included the fixed class effect of farm, and the Experiment 2. To assess the effect of sow parity random class effect of batch to account for possible en- on CL, gestation and litter characteristics, parity was vironmental variation. Statistical models for accuracy divided into 3 categories: class 1 (parities 2 and 3, n = of CL diameter measurements did not have farm in the 27), class 2 (parities 4 and 5, n = 35), and class 3 (pari- model since the measurements were done in for a sub- ties 6 to 9, n = 38) and the fixed class effect of parity set of sows coming from the same farm (n = 25). There (parity class 1, 2, and 3) was included in the model was no relationship between the parity of the sows and together with the random effect of batch. the CL measurements done by TUS and after ovarian To assess relationships between the measurements dissection (P > 0.05), so parity was not included in the of CL done by TUS and sow and litter characteristics , to- models. In all models, fixed class effects were corrected at birth, the continuous fixed effect of DIAM TUS with Bonferroni and if nonsignificant were removed gether with the fixed class effect of parity (1, 2, and 3) from the models. Interactions were never significant and the fixed class effect of litter size [class 1 (9 to 16 and were therefore removed from the models. piglets born, n = 34); class 2 (17 to 19 piglets born, n as as- First, the relationship between the OR = 36); and class 3 (20 to 26 piglets born, n = 30)] and TUS sessed by the 2 examiners separately (E1 and E2) was their interaction, were assessed on litter characteristics. investigated to check for inter-examiner agreement. In all models, batch was included as a random class ef- count- For this, the continuous linear effect of OR fect to account for possible environmental variation. If TUS counted by E1. ed by E2 was assessed on OR nonsignificant, the interactions and the fixed class ef- TUS Analyses on the accuracy of TUS were done using fects were removed from the models. Residuals from the data of E1 (n = 45). First, the difference between all models approximated normality based on skewness and OR in number of CL counted was cal- OR and kurtosis. Results are presented as the regression co- TUS DIS culated. Further, to check the accuracy of TUS in as- efficients (β) with their SE for continuous fixed effects relates with OR ) sessing OR (i.e., how does OR and as least squares means and their SE for fixed class TUS DIS was assessed on the continuous linear effect of OR effects. Results are considered significant at P ≤ 0.05. DIS . To check if the accuracy of TUS was affected OR TUS the continuous fixed effect of OR was by OR reSultS DIS DIS and OR . assessed on the difference between OR TUS DIS Regarding CL diameter measurements, the differ - Experiment 1 ence between average CL diameter estimated based on TUS and after ovarian dissection were assessed (i.e., The averages and SD of the CL, litter and sows – DIAM ). Further, to check the accuracy DIAM characteristics are presented in Table 1. In experiment TUS DIS of TUS in measuring the diameter of the 10 biggest 1, the total number of CL (mean ± SD) assessed by ) was 23.4 ± 5.8 (ranging from 13 to 34) CL, the continuous fixed effect of the average diam - TUS (OR TUS ) was eter of the 10 biggest CL at dissection (DIAM and the number counted after dissection of the ovaries DIS ) was 24.1 ± 5.3 (ranging from 13 to 37). The assessed on the average diameter of the 10 biggest CL (OR DIS ). To check if the accuracy of TUS by TUS (DIAM average diameter of the 10 biggest CL measured with TUS , the ) was 10.3 ± 0.7 mm and after dis- in measuring CL diameter was affected by OR TUS (DIAM DIS TUS was assessed on the ) was 10.3 ± 0.6 mm. The average continuous fixed effect of OR section (DIAM DIS DIS and DIAM . difference between DIAM diameter of all CL dissected was 9.8 ± 0.7 mm with an TUS DIS Also, to investigate the relationship between OR SD of 0.8 ± 0.4 mm. The average weight of the 10 big- ) was 0.40 ± 0.07 g, and the average and CL size, the continuous linear and quadratic effect gest CL (WT DIS was assessed on DIAM , DIAM and of OR weight of all CL dissected was 0.38 ± 0.07g. Average DIS TUS DIS ). on average CL weight after dissection (WT total luteal mass per sow was 7.9 ± 1.5g. DIS Moreover, aiming to check the relationship be- tween OR and characteristics of all CL dissected, the Relationship between CL Characteristics was continuous linear and quadratic effect of OR after Ovarian Dissection and with TUS DIS assessed on the average and standard deviation of CL diameter and weight of all CL, and on total luteal mass. Results show that there was a strong relationship assessed by the 2 examiners, E2 and Residuals of all models approximated normality between OR TUS based on skewness and kurtosis. Results are presented as E1 (β = 0.87 ± 0.09 CL E1/CL E2, P < 0.0001, R the regression coefficients (β) with their standard errors 0.93, Fig. 2A), showing that there was inter-observer (SE) for the continuous linear and quadratic fixed effects agreement in the assessment of OR using TUS. and as least square means and their SE for fixed class ef- Differences in CL number measured with TUS and fects. Results are considered significant at P ≤ 0.05. after ovarian dissection are presented in Fig. 3. The av- Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 511 Figure 2. Panel A, predicted relationship between OR assessed by TUS performed by examiner 2 and the OR assessed by TUS performed by exam- iner 1 [(n = 29), β = 0.87 ± 0.09 CLTUS E1/CLTUS E2, P < 0.0001, farm P = 0.12]. Panel B, predicted relationship between the OR assessed by E1 by TUS and OR after dissection [β = 0.28 ± 0.01 TUS/DIS, P = 0.01, farm P = 0.0002, (n = 45)]. Panel C; predicted relationship between the average diameter of the 10 biggest CL assessed after ovarian dissection and the average diameter of the 10 biggest CL assessed by TUS [(n = 25), β = 1.0 ± 0.07 mm TUS/ mm dissected, P < 0.0001]. Panel D, predicted relationship between the OR after dissection and the average CL diameter [TUS = 0.49 ± 0.19 (P = 0.02) – 0.01 ± 0.005 (P = 0.01) and DISS = 0.47 ± 0.18 (P = 0.02) – 0.01 ± 0.004 (P = 0.01)]. Statistical models included the fixed class effect of farm (n = 2, for panels a and b), and the random effect of batch (n = 6). Data points (♦) represent the predicted values estimated as the difference with the model residuals. sows the difference was 6 or more. So, there was not a close relationship between OR and OR [β = 0.28 DIS TUS ± 0.01 TUS/DISS, P = 0.01, farm 1 = 28.0 ± 1.3 and = 0.79; Fig. 2B]. farm 2 = 20.5 ± 1.2; P = 0.0002, R Furthermore, there was a positive linear relationship and the difference in OR assessed by between OR DIS TUS and after dissection (β = 0.72 ± 0.10 difference/CL = 0.68). So, dissected, P < 0.0001, farm P = 0.0002, R TUS is not an accurate method to estimate OR in sows at early pregnancy, and there is an increase in the inac- curacy of TUS with an increase in OR. Differences in the average CL diameter measured by TUS and the average CL diameter measured after – DIAM ) were as- ovarian dissection (DIAM Figure 3. Summary of the differences between the number of corpora TUS DIS lutea (CL) counted with transrectal ultrasonography (OR ) and the num- sessed. The average ± SD of the difference between the TUS ber of CL counted after slaughter and dissection of the ovaries (OR ) in DIS and DIAM was 0.02 ± 0.20 mm (rang- DIAM TUS DIS 45 multiparous sows at early pregnancy. Underestimations are shown in ing from –0.36 up to 0.34 mm difference). There was light gray and overestimations are shown in dark gray. a positive linear relationship between the DIAM DIS (β = 1.00 ± 0.07 mm TUS/mm DIS; and DIAM TUS erage ± SD of the difference in OR assessed by TUS = 0.96; Fig. 2C). Moreover, there was P < 0.0001, R and the difference be- and OR assessed after ovarian dissection was 0.7 ± 4.7 no relationship between OR DIS and DIAM (β = 0.005 ± 0.01 mm CL (ranging from –8 up to +12 CL difference). Results tween DIAM TUS DIS differed from = 0.44; P = 0.58). So, TUS show that in 24.4% of the sows OR difference/CL dissected, R TUS in by 0 or 1 CL; in 28.9% of the sows the dif- OR is an accurate method to estimate the average diameter DIS ference was 2 or 3, in 24.4% 4 or 5 and in 22.2% of the of the 10 biggest CL in sows in early pregnancy and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 512 Da Silva et al. the accuracy of the measurements done by TUS is not OR (P = 0.03)], which shows a maximum average DIS related with an increase in OR. CL diameter of 10.1 mm at 18 ovulations. There was and the and total Regarding the relationship between OR a negative linear relationship between OR DIS DIS average CL diameter and weight of the 10 biggest CL, average CL weight (β = - 0.01 ± 0.03 g/CL dissected, P and was not related with the SD there was a quadratic relationship between OR = 0.002). However, OR DIS DIS [0.49 ± 0.2 × OR (P = 0.02)– 0.01 ± the DIAM in CL diameter (P = 0.85) and with the SD in CL weight TUS DIS 2 2 (P = 0.01), R = 0.76; Fig. 2D] and 0.0004 × OR (P = 0.12). Furthermore, there was a positive linear re- DIS [0.47 ± 0.2 × OR (P = 0.02) – 0.01 ± 0.0004 and total luteal mass (β = + DIAM lationship between OR DIS DIS DIS 2 2 (P = 0.01), R = 0.69; Fig. 2D], which shows × OR 0.19 ± 0.1 g/CL dissected, P = 0.003). So, the total aver- DIS a maximum average diameter of the 10 biggest CL of age diameter and weight of the CL is lower in sows with 10.5 mm at 19 ovulations for measurements done with a higher OR, but there is no increase in variation in CL and DIAM , TUS and also after dissection (DIAM size with the increase in OR. Also, the total luteal mass TUS DIS respectively). Also, there was a negative linear relation- increases linearly with the increase in OR, despite the and the average weight of the 10 ship between OR decrease in average CL weight. DIS = 0.51; biggest CL (β = - 0.01 ± 0.003 g/CL dissected, R P = 0.01). So, the average diameter and weight of the 10 Experiment 2 biggest CL is lower in sows with a higher OR. There was a quadratic relationship between OR The averages and SD of sows, CL and litter char- DIS and the average diameter of all CL measured after dis- acteristics are presented in Table 1. The average CL (P = 0.05)– 0.01 ± 0.01 × section [0.43 ± 0.2 × OR diameter measured with TUS was 8.4 ± 0.8 mm, rang- DIS t able 1. Summary statistics of sows, corpus luteum and litter characteristics Variables n Mean SD Min Max Experiment 1 Parity 45 7.3 3.2 2 13 TUS pregnancy age, d 45 23.4 2.9 20 28 Slaughter pregnancy age, d 45 29.8 1.9 27 32 Ovulation rate by TUS 45 23.4 5.8 13 34 Ovulation rate after dissection 45 24.1 5.3 13 37 Average CL diameter TUS , mm 25 10.3 0.73 8.4 11.7 Average CL diameter DISS , mm 25 10.3 0.64 8.7 11.5 Average CL weight DISS , g 25 0.40 0.07 0.22 0.56 Total average CL diameter , mm 25 9.8 0.7 7.8 11.0 Total average CL weight , g 25 0.38 0.1 0.20 0.51 Total luteal mass , g 25 7.9 1.5 5.2 10.7 Experiment 2 Parity 100 5.0 1.9 2 9 TUS pregnancy age, d 100 23.8 2.4 21 29 Gestational length, d 100 115 1.7 111 120 Average CL diameter TUS , mm 100 8.4 0.8 5.5 10.5 Litter size 100 17.9 3.0 9 26 Number of live born 100 16.3 2.9 6 23 Number of stillborn 100 1.53 1.8 0 9 Number of mummies 100 0.42 0.8 0 5 Average piglet BW , g 100 1277 165 914 1618 SD BW , g 100 303 76 161 555 Average live born piglets BW , g 100 1299 167 933 1674 SD BW live born , g 100 292 76 115 492 Litter BW, kg 100 23 4.0 15 30 Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured by Transrectal ultrasonography (TUS). Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured with caliper rule after slaughter of the sows and ovarian dissection. Average weight of the 5 corpora lutea in each ovary (10 per sow) that had the highest diameter measured after ovarian dissection. Average diameter and weight calculated based on the measurement of all corpora lutea dissected from each ovary. Sum of the weight of all the corpora lutea dissected from the ovaries. Litter size is the sum of piglets born alive (live born) and stillborn piglets. Piglets were weighed within 24 h after birth. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 513 ing from 5.5 to 10.5 mm. The average total number of piglets born was 17.9 ± 3.0, and 16.3 ± 2.9 were born alive. The average birth weight (BW) of the total piglets born was 1,277 ± 165 g and the average within litter BW variation was 303 ± 76 g, the average BW of the piglets born alive was 1,299 ± 167 g and average within litter BW variation was 292 ± 76 g. Effect of Parity on Gestation Length, CL and Litter Characteristics Parity did not affect the gestation length, the aver - age diameter of the 10 biggest CL measured by TUS ), litter size, average and SD piglet birth (DIAM TUS weight or litter weight (P > 0.05). Sows in parity 6 up to 9 had a higher (2.13 ± 0.3, P = 0.02) number of stillborn piglets than sows in parity 2 up to 3 (0.86 ± 0.4) and 4 up to 5 (1.34 ± 0.3). Relationship between CL Diameter and Litter Characteristics and litter charac- Relationships between DIAM TUS teristics are presented in Table 2. There was a positive and average pig- linear relationship between DIAM TUS let BW [(β = + 37.6 ± 17.8 g/mm; P = 0.04, Fig. 4A) Figure 4. Panel A: estimated least square means for the effect of average + c value dependent on litter size (P < 0.0001): 9 to 16 CL diameter classes [5.5 to 7.8 mm (n = 23); 7.9 to 8.9 mm (n = 47); and 9.0 to 10.5 mm (n = 30)] on BW of total piglets born [P = 0.04; litter size class P piglets = 1367 ± 25 g, 17 to 19 piglets = 1,280 ± 24 g, < 0.0001]. Panel B: estimated least square means for the effect of CL diameter and 20 to 26 piglets = 1,171 ± 26 g]. There was also a classes on standard deviation (SD) of BW of the total piglets born (P = 0.02). and the positive linear relationship between DIAM TUS Statistical models included the fixed class effect of litter size [LS, 8 to 18 pig- SD in BW of the total born piglets (β = 24.3 ± 10.4 g/ lets born (n = 35); 17 to 19 piglets born (n = 36) and 20 to 26 piglets born (n = 30)] and its interactions, which were excluded from the models when not mm; P = 0.02, Fig. 4B) and the SD in BW of the piglets significant (P > 0.05). Significant differences between classes are indicated by born alive (β = 27.3 ± 10.5 g/mm, P = 0.01). There were letters above the columns and the error bars indicate the SE of the estimates. t able 2. Relationship between average corpora lutea diameter measured by transrectal ultrasonography (DIAM ) and litter characteristics at birth TUS DIAM , mm Litter size Parity TUS Dependent variables β (SEM) P-value P-value P-value Gestational length, d 0.48 (0.26) 0.06 ns ns Litter size –0.098 (0.43) 0.82 – ns Number of live born –0.084 (0.43) 0.84 – ns Number of stillborn –0.015 (0.22) 0.95 0.02 0.01 Number of mummies –0.013 (0.10) 0.90 ns ns Average piglet BW , g 37.57 (17.84) 0.04 < 0.0001 ns SD BW , g 24.25 (10.37) 0.02 ns ns Average live born piglets BW , g 32.98 (18.41) 0.08 < 0.0001 ns SD BW live born , g 27.33 (10.52) 0.01 ns ns Litter BW, kg 0.81 (0.47) 0.09 < 0.0001 ns Average calculated based on the diameter of the 5 biggest corpora lutea in each ovary (10 per sow) measured by TUS. ns = P > 0.05. Litter size is the sum of piglets born alive (live born) and stillborn piglets. Interaction between parity (PC) and litter size (LS) classes (P = 0.002): PC1 × LS1 (n = 9): 0.67 ± 0.55b; PC2 × LS1 (n = 12): 0.48 ± 0.48b; PC3 × LS1 (n = 6): 1.54 ± 0.47ab; PC2 × LS2 (n = 12): 1.02 ± 0.49b; PC2 × LS2 (n = 12): 2.01 ± 0.49b; PC3 × LS2 (n = 11): 0.92 ± 0.49ab; PC1 × LS3 (n = 13): 0.81 ± 0.66b; PC2 × LS3 (n = 12): 1.58 ± 0.51ab; PC3 × LS3 (n = 13): 3.83 ± 0.47a. Piglets were weighed within 24 h after birth (BW). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 514 Da Silva et al. no interactions between DIAM , litter size classes stroma, which makes visualization of each individual CL TUS and parity classes. So, an increase in the average CL more difficult. Moreover, despite the fact that transrectal diameter in early pregnancy is related with an increase ultrasonography is believed to provide clearer images of in average piglet birth weight and in within litter birth the ovaries due to less interference of intestinal tissues weight variation. (Kauffold and Althouse, 2007), is still possible that the ovaries were only partially visible due to interference of dIScuSSIon the intestinal and uterine tissue of the pregnant sows. Overestimations, on the other hand might be ex- This is, to our knowledge, the first study that in- plained by counting CL from part of an ovary 2 times, vestigated the accuracy of transrectal ultrasonography which might happen due to the proximity of the 2 ova- to assess CL number and diameter in sows in early ries during the examination and the difficulty in distin- pregnancy and investigated the relationship between guishing between the 2 separate ovaries. The lack of CL diameter measured by transrectal ultrasonography accuracy of transrectal ultrasonography in assessing the and litter characteristics at birth. number of CL in pregnant sows might also be related Transrectal ultrasonography did not provide an ac- with the high number of CL present in each ovary (high curate estimation of the number of corpora lutea (OR) ovulation rate), where crowding of the ovaries makes in sows in early pregnancy. The difference between the it more difficult to differentiate the CL. Gonzalez- OR assessed after ovarian dissection and with TUS was Añover et al. (2009) investigated the accuracy of tran- on average 0.7 ± 4.7 CL, ranging from an underestima- srectal ultrasonography in accessing the number of CL tion of 8 CL up to an overestimation of 12 CL. Moreover, in Iberian sows in the mid luteal phase (average ovu- only in 24.4% of the sows, the difference between OR lation rate of 6.0 ± 1.3), and achieved accuracy close assessed with TUS and after ovarian dissection was of to 100% in ovaries with 5 CL or less, which decreased only 1 CL, with 46.7% of the TUS estimations differing with the increase in number of CL present per ovary. with more than 4 CL. This inaccuracy occurred almost A decrease in accuracy with the increase in OR was equally due to under and over estimations of the number also observed by Soede et al. (1992) and Bolarin et al. of CL assessed after dissection of the ovaries (33.3 and (2009) when counting the number of pre-ovulatory fol- 42.2%, respectively). Underestimations are probably re- licles. In the present study (average OR of 24.1 ± 5.3) lated with the difficulty in visualizing all individual CL. there was also a decrease in accuracy of TUS (differ - Ultrasound machines uses high frequency sound waves ence between OR counted by TUS and after slaughter) and their echo to produce images (Pierson et al., 1988) with the increase in OR after ovarian dissection. This and different tissues have different abilities to reflect the could be related with the decrease in CL size with the sound waves (Pierson et al., 1988). Thus, characteristics increase in ovulation rate. Average CL diameter in sows of a tissue determine the proportion of the sound wave with 13 up to 22 ovulations was predicted to be 10.3 that will be reflected, which will then be represented on mm, decreasing to 8.7 mm in sows with more than 23 the ultrasound image display by dots of different shades ovulations. The same is true for the average CL weight, of gray, varying from black to white. Liquids, like fol- which can be 0.13g smaller in sows with more than 23 licular fluid, do not reflect sound waves (non-echogenic) ovulations in comparison with sows with 13 up to 22 and therefore the image appears as black on the screen, ovulations. A decrease in average CL weight (and thus in contrast with the surrounding ovarian tissue, that re- size, this study) with the increase in ovulation rate was flects part of the sound waves (echogenic) and can be also observed in gilts at 35 d of pregnancy (Da Silva seen as different shades of gray (Pierson et al., 1988). et al., 2017). This indicates that with a higher OR, not This contrast facilitates visualization of individual fol- only the ovaries are more crowded, but also individu- licles and explains the high accuracy of transrectal ul- al CL are smaller, which might increase the difficulty trasonography in assessing the number of pre-ovulatory in visualization of the individual CL, thus decreasing follicles in sows. Soede et al. (1992) described a differ - the accuracy of counting CL with TUS. The reason for ence of only 0.4 ± 1.8 between the number of follicles the decrease in CL size with the increase in ovulation counted by transrectal ultrasonography and the number rate is not known. During follicular growth, recruited of CL counted after slaughter of the sows (18.6 ± 3.5). follicles respond to FSH by increasing the production Also, Bolarin et al. (2009) observed a significant correla- of estradiol-17β (Britt and Findlay, 2002; Drummond, tion between the number of pre-ovulatory follicles (6 to 2006). The increase in estradiol-17β (E2) produc- 10 mm) counted per ovary with transrectal ultrasonogra- tion leads to an increase in granulosa cell number and phy and with laparoscopy (r = 0.98, P < 0.001). Corpora therefore to further follicular development (Drummond lutea, however, are tissue filled glands and are echogenic, and Findlay, 1999). So, together with the increase in surrounded by the also echogenic tissue of the ovarian E2 production, follicles increase in size. E2 production Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Corpora lutea and piglet birth weight 515 increases until it reaches a certain systemic threshold elongation is established by the conceptus (Geisert et al., concentration that triggers a pre-ovulatory GnRH/LH 2014) and more advanced embryos [derived from the surge that subsequently triggers ovulation (Drummond more developed oocytes, Pope et al. (1990)] elongate and Findlay, 1999; Drummond, 2006). With more fol- earlier and will have an increased uterine implantation licles recruited from the pool (i.e., higher OR), and length (Geisert et al., 1982a). The uterine implantation therefore more follicles producing E2, the threshold of length is related with the placental length of vital em- E2 necessary to trigger ovulation might occur when fol- bryos at 35 d of pregnancy in sows [β = 0.98 ± 0.14 cm/ licles are of smaller size than in sows with a lower OR. cm, P < 0.0001. C. L. A. Da Silva, unpublished results], Indeed, Knox et al. (2003) observed that systemic E2 which will possibly favor further fetal development and levels were the same at days –1 to +1 relative to the LH consequently piglet birth weight. peak (d 0) in gilts selected for high OR (OR 18.8 ± 0.4, Corpora lutea produce progesterone, which is of E2 37.9 ± 3.4 ng/mL) and in gilts of the control line (OR primary importance for maintenance of the pregnancy 14.3 ± 0.6, E2 44.7 ± 3.6 ng/mL), indicating that the in the pig (Spencer et al., 2004). So, it could be hypoth- threshold of E2 that precedes ovulation is independent esized that higher CL size in pregnant sows is related of OR. Soede et al. (1998) observed that the average with higher progesterone production favoring embry- volume of pre-ovulatory follicles at ovulation per sow onic growth and piglet birth weight. However, systemic was significantly correlated with the average CL weight progesterone levels in 238 gilts at 35 d of pregnancy (C. at 5 d of pregnancy (r = 0.28, P < 0.01), and Wientjes et L. A Da Silva, unpublished results), were not related al. (2012) observed that each mm increase in follicle di- with average CL weight (P = 0.69). It could also be pos- ameter at ovulation was related with 1.23 mm increase sible that average CL weight and piglet birth weight in CL diameter (P = 0.03) in multiparous sows at 10 d have a common origin in early pregnancy. Corpora lu- of pregnancy. Thus, high OR sows may have smaller tea regression occurs on d 15 to 16 of the oestrus cycle follicles at ovulation that develop into smaller CL. due to the increase in pulsatile endometrial secretion of We also investigated the validity of TUS to measure prostaglandin F2-ɑ (Moeljono et al., 1976). Thus, preg- CL diameter in sows. Results show that TUS is an ac- nancy recognition requires the development and main- curate method to assess CL diameter in sows at 3 to 4 wk tenance of CL beyond the luteal phase, and is a result of of pregnancy. There was a strong relationship between oestrogens (mainly estradiol-17β, E2) secretion by the the CL diameter measured by transrectal ultrasonogra- conceptuses on d 11 and 12, followed by a second peak phy and the CL diameter measured after dissection of between d 15 and 25 through 30 of pregnancy (Geisert the ovaries. Further, we investigated the relationship be- et al., 1990). Conceptuses E2 increases luteal LH re- tween average CL diameter assessed by transrectal ultra- ceptor concentration, and together with Il-β1, favors the sonography in early pregnancy and litter characteristics production of luteoprotective PGE2, which stimulates at birth and observed that there is an increase in average the expression of Vascular Endothelial Growth Factor piglet birth weight with an increase in average diameter (VEGF) in luteal cells, increasing luteal permeability of the 10 biggest CL. This might indicate that sows with and delivery of cholesterol to the luteal cells (Ziecik et a higher average CL diameters ovulated oocytes of better al., 2011), which might favor CL growth. So, concep- quality, that developed into embryos with higher growth tus development at the time of elongation and maternal potential and consequently into piglets with higher birth recognition of pregnancy may influence luteal vascu- weight. Indeed, in a recent study with 390 gilts slaugh- larization (through the effects of PGE2 and VEGF), tered at 35 d of pregnancy we found that there is an in- and thereby increase CL size. However, further inves- crease of 2.3 g in the weight of the vital embryos per tigations are needed to understand the mechanisms un- gram of increase in average CL weight [P = 0.001; C. derlying the relationship between CL size, embryonic L. A. Da Silva, unpublished results]. Heavier embryos growth and piglets birth weight. at d 35 of pregnancy might develop into heavier piglets An increase in average CL diameter was not only at birth. Larger/heavier CL develop from larger follicles related with average piglet birth weight as discussed at ovulation, as discussed above. Larger follicles at ovu- above, but also with an increase in within litter varia- lation are known to release oocytes with superior qual- tion in piglet birth weight. Within litter piglet birth ity due to a more advanced maturational status (Hunter, weight variation increases with the increase in litter size 2000; Gandolfi et al., 2005), which might lead to the (Milligan et al., 2002; Quiniou et al., 2002; Wolf et al., development of embryos with higher growth potential 2008), and is seen as a consequence of uterine crowding (Krisher, 2004). At d 11 and 12 of pregnancy, pig concep- on placental development in the early post implantation tuses transition from spherical to tubular and filamentous period (Foxcroft et al., 2006). However, in the current blastocysts in a process called elongation (Geisert et al., study, we did not observe a higher variation in piglet 1982a; Geisert et al., 1982b). Timing of rapid conceptus birth weight in bigger litters. 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Anim. 46:31–41. doi:10.1111/j.1439-0531.2011.01843.x APPendIX 1: lISt oF ABBreVIA tIonS Abbreviations Description TUS Transrectal ultrasonography OR Ovulation rate, i.e. the total number of CL present in both ovaries CL Corpora lutea OR Total number of corpora lutea counted on both ovaries with transrectal ultrasonography TUS E1 Transrectal ultrasonography examiner one E2 Transrectal ultrasonography examiner two DIAM The average diameter of the 10 biggest corpora lutea measured by transrectal ultrasonography in a sow TUS OR Total number of corpora lutea counted after slaughter and dissection of the ovaries for individual corpora lutea DIS DIAM The average diameter of the 10 biggest corpora lutea measured with a caliper rule after slaughter and dissection of the ovaries for DIS individual corpora lutea WT The average weight of the 10 biggest corpora lutea measured after slaughter and dissection of the ovaries for individual corpora lutea DIS LS Litter size, i.e. the sum of the number of piglets born alive and dead BW Piglet birth weight Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/4/507/4780407 by Ed 'DeepDyve' Gillespie user on 10 April 2018

Journal

Translational Animal Science – Oxford University Press

Published: Dec 1, 2017

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