Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Phenotypic and genetic relationships among anogenital distance, anti-Müllerian hormone, and in vitro embryo production in Gyr dairy cattle.

Anti-Müllerian hormone (AMH) concentration and number of recovered oocytes (ROOC) are phenotypic parameters associated with in vitro embryo production (IVEP). More recently, anogenital distance (AGD) has been proposed as a proxy for fertility in dairy cattle that is easy to collect at a low cost. The aim of this study was to characterize the AGD and its phenotypic and genetic associations with AMH and IVEP in Bos indicus Gyr dairy cattle. The hypothesis was that the number of ROOC, in vitro-produced embryos, and AMH concentration would increase as the AGD decreases. From July to December 2021, a single morphometrical measurement of AGD was collected in 552 donors from 6 herds in Brazil. A subset of donors had AMH assayed on the same day. Only ovum pick-up events that occurred up to 12 mo preceding and 7 mo succeeding the AGD measurement were used to assess the association between AGD, AMH, and IVEP. Thus, 472 donors (1,551 ovum pick-up events and 140 donors with AMH) were considered in the analysis. A raw average was calculated for each individual donor's ROOC, viable oocytes, total produced embryos, viability rate, and embryo rate (defined as total produced embryos/viable oocytes). Comparisons were conducted within the age categories of 3 to <6 yr or 6 to <10 yr. Phenotypic associations were performed in SAS software (SAS Institute Inc., Cary, NC). Genetic correlations were estimated using the BLUPF90 family of programs. The AGD (128.7 mm ± 14; mean ± standard deviation) had a normal distribution and was highly variable (83 to 172 mm) among the Gyr population. Our experimental hypothesis was partially supported by a phenotypic association of a greater number of total produced embryos (R2 = 0.023) as AGD decreased. Our results failed to support an increase in AMH concentration along with a decrease in AGD. In addition, positive and low genetic correlations were observed between AGD and viable oocytes (r = 0.08), and embryo rate (r = 0.20). A greater number of viable oocytes and embryos were observed in donors in the high compared with intermediate and low ROOC categories within both age categories. The age interval of 3 to <6 yr showed a greater number of recovered and viable oocytes for the high AMH compared with the low category, but no differences were observed among the AGD categories. In summary, for the Gyr breed, AGD was phenotypically inversely associated with a quantity-related parameter, such as the total number of produced embryos. In contrast, AGD showed a low genetic correlation with qualitative-related outcomes such as viable oocytes and embryo rate. Further studies should be performed to validate these retrospective analyses and to better understand the association between AGD and IVEP.

Genetic parameters for oocytes and embryo production and their association with linear type traits in dairy Gyr Cattle.

In vitro embryo production is one of the main reproductive techniques used in dairy Gyr cattle. In addition, linear type measures are well characterized and have been used in dairy Gyr breed selection for the last 4 decades. The estimation of genetic parameters for the number of aspirated oocytes and in vitro-produced embryos associated with the linear type measures would support genetic progress for animal breeding programs toward embryo production. This study aimed to estimate genetic parameters for aspirated oocytes, embryo in vitro production, and linear type traits, exploring the association between them. The repeatability model was applied to 14,251 ovum pick-up events from 1,916 Gyr donors. A subset of 604 donors from the same group had their body measurements taken. Single- and 2-trait analyses were carried out using the BLUPF90 family programs. Heritability estimates of 0.38, 0.34, and 0.20 were obtained for total oocytes, viable oocytes, and embryos, respectively,-and the heritability of the linear type traits ranged from 0.22 to 0.40. High genetic correlations between total oocytes and viable oocytes (0.99), and between oocytes (total and viable) and embryos (0.83) were obtained. Low to high genetic (-0.07 to 0.92) and phenotypic (0.32 to 0.86) correlations were obtained between the linear type traits. Moreover, low phenotypic correlations (0.01 to 0.13) were observed for oocytes (total and viable) and embryos with the linear type traits, whereas low to moderate genetic correlations (0.07 to 0.42) were observed between the same traits, especially for ilium width (0.42), rump area (0.38), and hip height (0.33). Thus, selection for in vitro production is achievable in Gyr dairy cattle, and superior genetic progress is associated with the selection of oocytes (total and viable). Furthermore, the moderate genetic association between oocytes and embryos with linear type traits, especially ilium width suggests that progress on in vitro embryo production may be achieved by accessing these measurements.

Temporality of ovarian steroids and LH/FSH pulse profiles encompassing selection of the dominant follicle in heifers†.

The tested hypotheses were (1) LH/FSH pulses and F2 diameter are diminished by P4 and, (2) E2 increases during the transition to deviation and alters LH/FSH pulses. On Day 5 (Day 0 = ovulation), heifers were randomized into an untreated group (HiP4, n = 11), and a prostaglandin analog treated group (NoP4, n = 10). On Day 6, a follicular wave was induced by follicle ablation. Ultrasound and blood collections were performed every 12 h from Days 7 to 11. Blood was collected every 15 min for 10 h on Day 9 (largest follicle expected to be ~7.5 mm). Estradiol was ~75% greater (0.36 ± 0.14 vs 0.63 ± 0.19 pg/mL) in heifers with F1 ≥ 7.2 mm than in heifers with F1 < 7.2 mm. The HiP4 had smaller second largest follicle (F2) diameter, lower estradiol (P = 0.06), LH pulse baseline and peak concentrations (P < 0.007), in addition to half the frequency of LH/FSH pulses (4.1 ± 0.3 vs 9.6 ± 0.7 in 10 h) than the NoP4. Within HiP4, heifers with F1 ≥ 7.2 mm had ~25% fewer (P = 0.03) LH pulses compared to heifers with F1 < 7.2 mm. In contrast, within the NoP4, heifers with F1 ≥ 7.2 mm had ~75% greater LH (P = 0.05) and FSH (P = 0.08) pulse amplitude. We propose that greater F2 diameter at deviation in low P4 is related to greater LH baseline and peak concentrations, and greater frequency of LH/FSH pulses. A greater increase in E2 after F1 reaches ~7.2 mm results in further stimulation of LH/FSH pulse amplitude. Elevated P4 not only diminished frequency of LH/FSH pulses but also converted an E2 increase into a negative feedback effect on LH/FSH pulse frequency leading to smaller F2 at deviation.

Processes involved in prostaglandin F2alpha autoamplification in heifers.

In brief: Endometrial and luteal synthesis of prostaglandin F2alpha (PGF2A) occurs before and during luteolysis and is critical for luteal regression. This study demonstrates that PGF2A stimulates further PGF2A synthesis (autoamplification) apparently from the corpus luteum. Abstract: Understanding the endocrine profile of prostaglandin F2alpha (PGF2A) autoamplification is fundamental to comprehend luteal and endometrial responses to PGF2A. On day 10 of postovulation (preluteolysis), heifers (n = 6/group) were treated intrauterine with saline or PGF2A (0.5 mg; hour 0). A third group received flunixin meglumine + PGF (FM+PGF) to prevent endogenous synthesis of PGF2A. Exogenous PGF2A was metabolized at hour 2 as measured by PGF2A metabolite (PGFM). From hours 5 to 48, concentrations of PGFM were greatest in the PGF group, smallest in the FM+PGF, and intermediate in the control suggesting endogenous synthesis of PGF2A only in PGF group. Progesterone (P4) concentrations decreased transiently between hours 0 and 1 in PGF and FM+PGF groups but rebounded to pretreatment concentrations by hours 6 and 4, respectively. No control or FM+PGF heifers underwent luteolysis during the experimental period. Conversely, in the PGF group, one heifer had complete luteolysis (P4 < 1 ng/mL), two heifers had partial luteolysis followed by P4 and CL resurgence by hour 48, and three heifers did not undergo luteolysis. Endogenous PGF2A appears to be of luteal origin due to the lack of pulsatile pattern of PGFM and lack of endometrial upregulation of oxytocin receptor (typical of endometrial synthesis of PGF2A), whereas luteal downregulation of PGF receptor and HPGD indicates a classic luteal response to PGF2A signaling although other specific mechanisms were not investigated. The hypothesis was supported that a single PGF2A treatment simulating the peak of a natural luteolytic pulse and the uteroovarian transport of PGF2A stimulates measurable endogenous PGF2A production.

SMAD6 inhibits granulosa cell proliferation and follicle growth rate in carrier and noncarrier heifers of the Trio allele.

In brief: Follicle selection is a key event in monovular species. In this manuscript, we demonstrate the role of SMAD6 in promoting decreased granulosa cell proliferation and follicle growth rate in carriers vs noncarriers of the Trio allele and after vs before follicle deviation. Abstract: Cattle are generally considered a monovular species; however, recently, a bovine high fecundity allele, termed the Trio allele, was discovered. Carriers of Trio have an elevated ovulation rate (3-5), while half-sibling noncarriers are monovular. Carriers of the Trio allele have overexpression in granulosa cells of SMAD6, an inhibitor of oocyte-derived regulators of granulosa cell proliferation and differentiation. In experiment 1, follicle size was tracked for each follicle during a follicular wave. Follicle growth rate was greater before vs after follicle deviation in both carriers and noncarriers. Additionally, follicle growth rate was consistently less in carriers vs noncarriers. In experiment 2, we collected granulosa cells from follicles before and after deviation for evaluation of granulosa cell gene expression. Granulosa cell proliferation was less in carriers vs noncarriers and after vs before follicle deviation (decreased expression of cell cycle genes CCNB1 and CCNA2). The decreased granulosa cell proliferation in noncarriers after deviation was associated with increased SMAD6 expression. Similarly, in experiment 3, decreased expression of SMAD6 in granulosa cells of noncarriers cultured in vitro for 60 h was associated with increased expression of cell cycle genes. This suggests that SMAD6 may not just be inhibiting follicle growth rate in carriers of Trio but may also play a role in the decreased follicle growth after deviation in noncarriers. The hypotheses were supported that (1) follicle growth and granulosa cell proliferation decrease after deviation in both carriers and noncarriers and that (2) granulosa cell proliferation is reduced in carriers compared to noncarriers.

Calving date as a potential predictor for the probability of approval in the first breeding soundness evaluation of Nellore bulls.

Beef production systems primarily use natural service (NS) for breeding. However, a significant number of bulls used for NS are subfertile, limiting the profitability of the cow-calf operations. Therefore, producers should select bulls based on breeding soundness evolutions (BSE) to ensure higher pregnancy rates. Several factors can affect the bull ability to pass a BSE. We hypothesize that calving date would be a factor that affects the bull probability of approval at the first BSE. For this purpose, a multivariate logistic regression in a dataset of 14,737 BSEs from young Nellore bulls was used. Correlations between calving date, biometrics, and semen traits were evaluated using Pearson's correlation coefficient. Our results demonstrated that the calving date affected the probability of approval at the first BSE (p < .05). Indeed, the variable that added more information to our model was the calving date, far more than the age group of the bulls according to Akaike's information criterion. Hence, bulls born on day 0 of the calving season have 1.26 more chances to be approved at the first BSE than bulls born 21 days later. This result highlights the importance of getting the dams of future bulls pregnant as soon as possible in the breeding season. In addition, the calving season should be no longer than 47 days to achieve 80% BSE approval in 20-22 months old Nellore bulls. The strongest correlation was with SC, which decreased as the calving date increased. Therefore, the calving date may be used as a predictor of the outcome of the first BSE in young bulls. In that way, the calving date can help seedstock producers to maximize efficiency in making crucial management decisions during the breeding and calving season including nutrition, reproductive, and culling.

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5 mg, respectively) during mid-luteal phase (12 and 10 days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4 h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation, follicle dynamics, and circulating follicle-stimulating hormone in lactating dairy cows.

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 µg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (∼18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5-11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.

Up-regulation of endometrial oxytocin receptor is associated with the timing of luteolysis in heifers with two and three follicular waves†.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation = day 0), Holstein heifers were randomized into two groups: cytobrush group (n = 9) had an endometrial sample collected every 48 h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n = 4) or at the end of luteolysis (n = 5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.