Review: The ovarian follicular reserve - implications for fertility in ruminants.

Ruminants are born with a finite number of healthy ovarian follicles and oocytes (ovarian reserve) and germ cell proliferation in the developing foetal gonad predominantly occurs during early gestation. Two markers have been established to reliably estimate the size of the ovarian reserve in cattle: the number of antral follicles ≤3 mm in diameter recruited per follicular wave (Antral Follicle Count, AFC) and peripheral concentrations of the Anti-Müllerian hormone (AMH). Studies that used one or both indicators show that the size of ovarian reserve varies greatly among age-matched individuals, but is highly repeatable in the same animal. Conditions during prenatal life are likely among the causes of such variation in the ovarian reserve. In addition, the size of the ovarian reserve is a moderately heritable trait in cattle. The association between ovarian reserve and fertility is controversial. Several studies indicate that cattle with a low ovarian reserve have phenotypic characteristics that are associated with suboptimal fertility. On the contrary, the presence and absence of a positive association between AFC and/or AMH and fertility measures (i.e. no. on services/conception, pregnancy rates, pregnancy loss) have been equally reported in cattle. In conclusion, the size of the ovarian reserve in the progeny can be enhanced by improving management of the dam from preconception to early gestation and also through genetic selection. However, although the ovarian reserve may be among the determinants of reproductive success in ruminants, the use of AFC/AMH as reliable predictors of fertility is yet to be established. Furthermore, the possibility that there is a complex interaction of AFC, AMH and reproduction has yet to be fully characterised and exploited to improve fertility in cattle.

Supplementation with Yucca schidigera improves antioxidant capability and immune function and decreases fecal score of dairy calves before weaning.

Yucca schidigera (YS) is a species of plant rich in antimicrobials, antioxidants, and immunomodulators. It has been used as feed additive to improve animal performance and decrease methane emissions in cattle. However, few studies have evaluated YS in dairy calves. In this study, we evaluated the effects of YS on the growth performance, antioxidant capacity, and immune function in dairy calves before weaning. We randomly assigned 40 newborn female Holstein calves (4 d old; 40 ± 5 kg of body weight) to 1 of 4 treatments (n = 10 per treatment), which were fed 0, 3, 6, or 9 g/d of YS powder. The YS allowance was mixed into milk or milk replacer and fed twice daily. Dry matter intake (both liquid and starter feed) and fecal score were recorded daily, and body weight, withers height, body length, and heart girth were measured at 4, 14, 28, 42, and 60 d of age. Blood was sampled from the jugular vein at 14, 42, and 60 d of age after the afternoon feeding for analysis of serum antioxidant capacity and immune function. Feeding YS did not affect dry matter intake, but decreased the feed-to-gain ratio with a quadratic dose effect. Over the whole study period, the average daily gain tended to linearly increase with the increasing YS doses, and it was 6.8% higher in diets supplemented with 9 g/d of YS than in the basal control diet without YS. The YS supplementation linearly decreased fecal score in a dose-dependent manner, and the frequency of diarrhea was significantly decreased as the YS supplementation increased throughout the whole study period. The YS supplementation also linearly decreased maleic dialdehyde concentration in the serum compared with the control group. The activity of catalase tended to linearly and quadratically increase, and that of glutathione peroxidase increased linearly with the increased YS supplementation. Serum concentrations of IgA and IgG increased linearly with the increased YS supplementation, and that of IgG tended to increase quadratically. To the best of our knowledge, this is the first study that demonstrated that feeding YS to young calves could improve growth, feed efficiency, and immunity, and decrease fecal score and diarrhea. The results of this study indicated that feeding YS at 9 g/d may be recommended to benefit dairy calves before weaning.

Exposure of dairy cows to high environmental temperatures and their lactation status impairs establishment of the ovarian reserve in their offspring.

The objectives of this study were to establish if exposure of pregnant dairy cows to high environmental temperatures and humidity during the first trimester of pregnancy impairs the establishment of the ovarian reserve (total number of healthy follicles and oocytes in ovaries) and fertility in their offspring. Serum anti-Müllerian hormone (AMH) concentrations and number of follicles ≥3 mm (antral follicle count; AFC) were assessed on a random day of the estrous cycle in 310 sixteen-month-old dairy heifers. Based on season of their conception and early fetal life, heifers were separated into 2 groups: summer (mean monthly temperature-humidity index = 69.33 ± 2.6) and winter (temperature-humidity index = 54.91 ± 1.08). The AMH and AFC were lower in summer (419.27 ± 22.81 pg/mL and 9.32 ± 0.42 follicles, respectively) compared with winter heifers (634.91 ± 47.60 pg/mL and 11.84 ± 0.46 follicles, respectively) and were not influenced by farm and age at sampling. Heifers born to dams that were not being milked during gestation had lower AMH and AFC compared with offspring of cows on their first lactation, whereas no difference was detected between offspring of cows on their first and subsequent lactations. Summer and winter heifers had similar age at first service and at first calving, and similar number of services per conception. Regardless of season in early fetal life, heifers were classified into 3 groups based on AMH and AFC (low = 20%, intermediate = 60%, high = 20%). Heifers with the lowest AMH were older at first service compared with herd mates with intermediate AMH, but age at first calving and number of services per conception were similar among AMH categories. No difference was detected in any of the fertility measures among AFC categories. Heifers born to mothers exposed to high environmental temperatures in early gestation had smaller ovarian reserves compared with herd mates conceived in winter, but no association between season of early fetal life and fertility at first conception was established. Season of conception and maternal lactation status affect the size of the ovarian reserve, but not fertility, at first conception in the progeny.

Cervical mucus sialic acid content determines the ability of frozen-thawed ram sperm to migrate through the cervix.

The aim of this study was to investigate the properties and to functionally characterize the cervical mucus that modulates sperm transport through the cervix by using ewe breeds with a divergent pregnancy rate (Belclare and Suffolk; high and low, respectively) following cervical insemination using frozen-thawed semen. Sperm number, as well as sialic acid and fucose content in both the channels and in the lumen of different regions of the cervix were quantified in inseminated Belclare and Suffolk ewes. Expression of glycosyltransferase and MUC genes, glycosidase activity and sialic acid speciation in follicular phase cervical tissue and mucus were assessed. More spermatozoa were found in the cervical channels in the region closest to the cervical os in Belclare than Suffolk ewes (P < 0.05) and Suffolk ewes had a higher sialic acid content in the cervical channels than Belclare ewes (P < 0.05) in all regions of cervix. Suffolk ewes had significantly higher expression of FUT1, ST6GAL1 and MUC5AC than Belclare ewes. There was no difference between the breeds in glycosidase activity (P > 0.05). Levels of Neu5Ac were higher in Belclare than Suffolk ewes (P < 0.05) and levels of Neu5Gc was higher in Suffolk than Belclare ewes (P < 0.05). Competitive sperm penetration assays demonstrated that frozen-thawed sperm progression increased when cervical mucus was incubated with sialyllactose prior to a sperm penetration test (P < 0.05). These results suggest that the difference between Belclare and Suffolk ewes in sperm transport with frozen-thawed semen is due to the higher concentration of sialic acid within channels, which binds to spermatozoa and reduces their ability to traverse the cervix.

Siglec expression on the surface of human, bull and ram sperm.

Sialic acid (Sia) is a major constituent of both the sperm glycocalyx and female reproductive mucosal surface and is involved in regulating sperm migration, uterotubal reservoir formation and oocyte binding. Siglecs (sialic acid-binding immunoglobulin - like lectins) commonly found on immune cells, bind to Sia in a linkage- and sugar-specific manner and often mediate cell-to-cell interactions and signalling. Proteomic and transcriptomic analysis of human and bovine sperm have listed Siglecs, but to date, their presence and/or localisation on sperm has not been studied. Therefore, the aim of this study was to characterise the presence of Siglecs on the surface of bovine, human and ovine sperm using both immunostaining and Western blotting. Siglec 1, 2, 5, 6, 10 and 14 were identified and displayed both species- and regional-specific expression on sperm. Almost universal expression across Siglecs and species was evident in the sperm neck and midpiece region while variable expression among Siglecs, similar among species, was detected in the head and tail regions of the sperm. The possible role for these proteins on sperm is discussed.

Establishment of critical timing of progesterone supplementation on corpus luteum and embryo development in beef heifers.

Optimal concentrations of progesterone (P4) during early pregnancy are a major determinant of embryo survival in cattle. This study examined the effects of P4 supplementation, following a period of induced low P4, on corpus luteum (CL) development, circulating P4 concentrations and embryo development. A total of 107 beef heifers from one herd were used in the study. Following AI (Day 0) at a synchronized oestrus, heifers were randomly assigned to 1 of 5 treatment groups: (1) no subsequent treatment (control; n=15); (2) administration of a synthetic prostaglandin F2α analogue (PG) on Days 3, 3.5 and 4 to induce low P4 and no further treatment (LP4; n=23); administration of PG as above followed by P4 supplementation via insertion of a Controlled Internal Drug Release device from (3) Day 4-7 (P4 d4-7; n=22); (4) Day 4-10 (P4 d4-10; n=23); or (5) Day 7-10 (P4 d7-10; n=24). Embryo and CL characteristics were determined following slaughter on Day 16. Embryo size was greater in heifers administered P4 d4-7 and P4 d4-10 in comparison with LP4. Corpus luteum area was significantly reduced in heifers administered P4 d4-7 and P4 d4-10 up to Day 7, and Day 10 (P4 d4-10), in comparison with LP4 heifers. Initiation of supplemental P4 on Day 7 had no effect on CL area or embryo size in comparison with LP4 heifers. Concentrations of P4 were less in P4 d4-7, P4 d7-10 and P4 d4-10 on Day 15 in comparison with LP4 heifers. The results of the study indicate an initiation and duration effect of supplemental P4 on circulating P4, embryo and CL development following induction of LP4 on Day 3 and 4.

Anti-Müllerian Hormone (AMH) and fertility management in agricultural species.

A reliable, easy to assess marker for fertility in agricultural species would be highly desirable and Anti-Müllerian Hormone (AMH) is a promising candidate. This review summarizes recent findings concerning AMH and its role in fertility management, mainly in cattle. It focuses on (1) alterations in circulating AMH concentrations from birth to puberty and during estrous cycles; (2) correlation of circulating AMH concentrations with ovarian follicle numbers and ovarian reserve; (3) factors that impact circulating AMH concentrations; (4) use of AMH as a predictor of fertility. Circulating AMH concentrations can be easily and reliably measured with a single blood sample in adult cattle because AMH varies minimally during the estrous cycle and is repeatable across multiple cycles. Circulating AMH concentrations are positively associated with several measures of fertility. Dairy heifers with low compared with higher AMH concentrations subsequently had lower pregnancy rates, higher probability of being culled after birth of their first calf and shorter herd longevity. Also, AMH is predictive of response to superovulation in cattle and sheep. Several factors contribute to the variability in AMH concentrations among individuals; for example, beef cattle have higher AMH than dairy cattle. Nutritional imbalances, disease and endocrine disruptors during fetal life may negatively program the size of the ovarian reserve and consequently serum AMH concentrations and potential fertility in adulthood. We conclude that AMH may be a predictor of fertility and herd longevity in cattle, whereas in sheep and other farm species, the potential association between AMH and reproductive performance remains largely unexplored.Free Italian abstract: An Italian translation of this abstract is freely available at http://www.reproduction-online.org/content/154/1/R1/suppl/DC1.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

The concurrent and carry over effects of long term changes in energy intake before insemination on pregnancy per artificial insemination in heifers.

Follicle development in a period of negative energy balance (NEB), as experienced by the postpartum dairy cow, could be affected by undesirable metabolic changes, and may contain a developmentally incompetent oocyte with an impaired potential to establish a pregnancy. A differential feeding model in heifers was developed to evaluate the concurrent and carryover effects of reduced energy intake before insemination on pregnancy per artificial insemination (P/AI). Heifers were randomly assigned to either (i) control feed intake group (CF), n=68, 1.3 times estimated maintenance energy (M) requirements for 50 days and 2.0 M for 83 days or (ii) restricted feed intake (RF), n=88, 0.65 M for 50 days and 2.0 M for 83 days. Pregnancy per AI was determined by transrectal ultrasonography at day 30 following AI. Despite significant loss of live weight (LW; 5.8±2.1 vs 70.5±2.8 kg, respectively) and body condition score (BCS; 0.05±0.03 vs 0.45±0.03) and a significant elevation in systemic concentrations of non-esterified fatty acids in RF heifers, there was no concurrent effect on P/AI (69 vs 72%) following AI at day 50. However, there was a carryover effect on P/AI as there was an 18 percentage point difference (64 vs 82%) between CF and RF heifers following AI on day 93. The results of the study indicate that a reduction in energy intake for a 50-day period pre-insemination had no concurrent effect but had a positive carryover effect on P/AI.

Evidence of a molecular clock in the ovine ovary and the influence of photoperiod.

The influence of the central circadian clock on reproductive timing is well established. Much less is known about the role of peripheral oscillators such as those in the ovary. We investigated the influence of photoperiod and timing of the LH surge on expression of circadian clock genes and genes involved in steroidogenesis in ovine ovarian stroma. Seventy-two Suffolk cross ewes were divided into two groups, and their estrous cycles were synchronized. Progestagen sponge removal was staggered by 12 hours between the groups such that expected LH peak would occur midway through either the light or dark phase of the photoperiodic cycle. Four animals from each group were killed, and their ovaries were harvested beginning 36 hours after sponge removal, at 6-hour intervals for 48 hours. Blood was sampled every 3 hours for the period 24 to 48 hours after sponge removal to detect the LH surge. The interval to peak LH did not differ between the groups (36.2 ± 1.2 and 35.6 ± 1.1 hours, respectively). There was an interaction between group and the time of sponge removal on the expression of the core clock genes ARNTL, PER1, CRY1, CLOCK, and DBP (P < 0.01, P < 0.05, P < 0.01, P < 0.01, and P < 0.01, respectively). As no significant interaction between group and time of day was detected, the datasets were combined. Statistically significant rhythmic oscillation was observed for ARNTL, CLOCK, CRY1 (P < 0.01, respectively), PTGS2, DBP, PTGER2, and CYP17A1 (P < 0.05, respectively), confirming the existence of a time-sensitive functionality within the ovary, which may influence steroidogenesis and is independent of the ovulatory cycle.

Concentration of anti-Müllerian hormone in dairy heifers is positively associated with productive herd life.

Reliable biomarkers predictive of productive herd life (time in herd after birth of first calf) have heretofore not been discovered in dairy cattle. However, circulating concentrations of anti-Müllerian hormone (AMH) are positively associated with number of follicles or antral follicle count (AFC), ovarian function, and fertility, and approximately 25% of cows have a relatively low AFC and low AMH concentrations. The present study tested the hypothesis that heifers with the lowest AMH concentrations have suboptimal fertility and are removed from a herd for poor reproductive performance at a greater rate, and therefore have a shorter productive herd life compared with age-matched herdmates with higher AMH. To test this hypothesis, 11- to 15-mo-old Holstein heifers (n=281) were subjected to a single measurement of AMH. All heifers not removed from the herd had the opportunity to complete 2 lactations and start their third lactation after calving. During this time, performance and health parameters for each individual were recorded daily by herd managers. Results showed that the quartile of heifers with the lowest AMH concentration also had, on average, a shorter productive herd life (by 196 d), a reduced survival rate after birth of the first calf, the lowest level of milk production (first lactation), the lowest total percentage of cows pregnant (across all lactations), the highest culling rates (first and second lactations and overall), and the highest culling rate for poor reproduction (first lactation) compared with age-matched herdmates with higher AMH. We concluded that a single determination of AMH concentration in young adult dairy heifers may be a simple diagnostic method to predict herd longevity, and AMH may be a useful phenotypic marker to improve longevity of dairy cows.

Ultrasound monitoring of blood flow and echotexture of the corpus luteum and uterus during early pregnancy of beef heifers.

The aim was to characterize changes in the ultrasound characteristics of the CL and uterus in pregnant, inseminated nonpregnant, and cyclic beef heifers and to correlate findings with systemic progesterone (P4) concentrations with the intention of identifying possible markers for early identification of pregnancy. Heifers were randomly selected for artificial insemination after estrus synchronization. Ultrasound examinations of the CL and uterus were carried out by transrectal ultrasonography using a high-resolution ultrasound scanner equipped with a 12 MHz linear array probe on Days 7, 11, 14, 16, and 18 after artificial insemination (Day 0; i.e., estrus). Cross-sectional B-mode images of the CL were captured for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow were captured and stored for analysis of luteal blood flow area and ratio. Longitudinal B-mode images of the uterine horns were captured just beyond the bifurcation of the uterine horns and stored for analysis of contrast and homogeneity (MaZda v4.6; Technical University of Lodz, Institute of Electronics, Poland). A total of three images were captured for each structure of interest. Serum concentrations of P4 were determined from blood samples collected at each ultrasound examination. After pregnancy diagnosis by ultrasound, heifers were retrospectively allocated as being pregnant (embryonic heartbeat on Day 28; n = 14) or nonpregnant (interestrous interval 18-21 days; n = 8) and their data were compared with noninseminated cyclic heifers (n = 10). Corpus luteum tissue area did not appear to change between pregnant, nonpregnant, or cyclic control groups between Days 7 and 18 (P > 0.05). No significant differences in CL echotexture characteristics were found between groups at any time point. There were no significant differences between pregnant, nonpregnant, and cyclic control groups for CL blood flow area (P > 0.05). However, CL blood flow ratio decreased significantly (P < 0.05) in both inseminated nonpregnant and cyclic heifers between Days 14 and 18, whereas it remained unchanged in pregnant heifers (P > 0.05). Uterine homogeneity was not significantly different between groups at any time point (P > 0.05). However, uterine contrast was significantly greater (P < 0.05) in pregnant compared with cyclic control heifers on Days 16 and 18. Concentrations of P4 were lower (P < 0.05) in nonpregnant and control heifers than in pregnant heifers from Days 16 to 18. In conclusion, there were differences between nonpregnant and cyclic heifers compared with pregnant heifers in P4 concentrations from Day 16. On Day 18, the CL and uterine characteristics were different between the nonpregnant and pregnant heifers. Ultrasound measures of CL blood flow and uterine echotexture may be useful to establish pregnancy status. Further investigation is required to identify if pregnancy diagnosis can be made on Day 18 or at a later day postpartum.

N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages.

Follicular fluid (FF), an important microenvironment for the development of oocytes, contains many proteins that are glycosylated with N-linked glycans. This study aimed i) to present an initial analysis of the N-linked glycan profile of bovine FF using hydrophilic interaction liquid chromatography, anion exchange chromatography, high performance liquid chromatography (HPLC)-based separations and subsequent liquid chromatography-mass spectrometry/mass spectrometry analysis; ii) to determine differences in the N-glycan profile between FF from dominant and subordinate follicles from dairy heifers and lactating dairy cows and iii) to identify alterations in the N-glycan profile of FF during preovulatory follicle development using newly selected, differentiated (preovulatory) and luteinised dominant follicles from dairy heifers and lactating cows. We found that the majority of glycans on bovine FF are based on biantennary hypersialylated structures, where the glycans are sialylated on both the galactose and N-acetylglucosamine terminal sugars. A comparison of FF N-glycans from cows and heifers indicated higher levels of nonsialylated glycans with a lower proportion of sialylated glycans in cows than in heifers. Overall, as the follicle develops from Selection, Differentiation and Luteinisation in both cows and heifers, there is an overall decrease in sialylated structures on FF N-glycans.

Effect of exogenous progesterone supplementation in the early luteal phase post-insemination on pregnancy per artificial insemination in Holstein-Friesian cows.

One of the main determining factors of pregnancy per artificial insemination (P/AI) is an optimum concentration of progesterone (P4) in the early luteal phase. This study examined the effects of P4 supplementation on P/AI in lactating Holstein-Friesian cows. A total of 453 cows in 8 spring-calving herds were used in the study. Following AI, cows were randomly assigned to 1 of 2 treatment groups: (1) no subsequent treatment (control; n=221); (2) insertion of a Controlled Internal Drug Release device (CIDR) from day 4 to day 9 post-estrus (supplemented; n=232). Pregnancy per AI was determined by transrectal ultrasonography at day 30 following AI. Insertion of a CIDR increased concentrations of milk P4 in supplemented cows by 4.78ng/mL between day 4 and 4.5 in comparison with a 0.55ng/mL increase in control cows. Progesterone supplementation from day 4 to 9 after AI decreased P/AI by 12 percentage points (56 vs 44%). There was a positive linear and quadratic relationship between P/AI and milk concentration of P4 on day 4 post-estrus in control cows. An optimum concentration of 2.5ng/mL on day 4 was calculated from the logistic regression curve to achieve a probability of P/AI of 65%. When both treatments groups were included in the analysis, there was no association between P/AI and concentrations of P4 on day 4. The results of the study indicate that supplementation with P4 initiated in the early luteal phase had a negative effect on P/AI in dairy cows.

Expression of microRNAs and their target genes and pathways associated with ovarian follicle development in cattle.

Development of ovarian follicles is controlled at the molecular level by several gene products whose precise expression leads to regression or ovulation of follicles. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through sequence-specific base pairing with target messenger RNAs (mRNAs) causing translation repression or mRNA degradation. The aim of this study was to identify miRNAs expressed in theca and/or granulosa layers and their putative target genes/pathways that are involved in bovine ovarian follicle development. By using miRCURY microarray (Exiqon) we identified 14 and 49 differentially expressed miRNAs (P < 0.01) between dominant and subordinate follicles in theca and granulosa cells, respectively. The expression levels of four selected miRNAs were confirmed by qRT-PCR. To identify target prediction and pathways of differentially expressed miRNAs we used Union of Genes option in DIANA miRPath v.2.0 software. The predicted targets for these miRNAs were enriched for pathways involving oocyte meiosis, Wnt, TGF-beta, ErbB, insulin, P13K-Akt, and MAPK signaling pathways. This study identified differentially expressed miRNAs in the theca and granulosa cells of dominant and subordinate follicles and implicates them in having important roles in regulating known molecular pathways that determine the fate of ovarian follicle development.

Heritability and impact of environmental effects during pregnancy on antral follicle count in cattle.

Previous studies have documented that ovarian antral follicle count (AFC) is positively correlated with number of healthy follicles and oocytes in ovaries (ovarian reserve), as well as ovarian function and fertility in cattle. However, environmental factors (e.g., nutrition, steroids) during pregnancy in cattle and sheep can reduce AFC in offspring. The role that genetic and environmental factors play in influencing the variability in AFC and, correspondingly, the size of the ovarian reserve, ovarian function, and fertility, are, however, poorly understood. The present study tests the hypothesis that variability in AFC in offspring is influenced not only by genetic merit but also by the dam age and lactation status (lactating cows vs. nonlactating heifers) and milk production during pregnancy. Antral follicle count was assessed by ultrasonography in 445 Irish Holstein-Friesian dairy cows and 522 US Holstein-Friesian dairy heifers. Heritability estimates for AFC (± standard error) were 0.31 ± 0.14 and 0.25 ± 0.13 in dairy cows and heifers, respectively. Association analysis between both genotypic sire data and phenotypic dam data with AFC in their daughters was performed using regression and generalized linear models. Antral follicle count was negatively associated with genetic merit for milk fat concentration. Also, AFC was greater in offspring of dams that were lactating (n=255) compared with nonlactating dams (n=89) during pregnancy and was positively associated with dam milk fat concentration and milk fat-to-protein ratio. In conclusion, AFC in dairy cattle is a moderately heritable genetic trait affected by age or lactation status and milk quality but not by level of dam's milk production during pregnancy.

Early pregnancy diagnosis on days 18 to 21 postinsemination using high-resolution imaging in lactating dairy cows.

The aim was to assess the ability of corpus luteum (CL) and uterine ultrasound characteristics on d 18 to 21 to predict pregnancy status in lactating dairy cows. Ultrasound examinations were carried out on cows (n = 164) on d 18 to 21 following artificial insemination (AI). Images of the uterus and CL were captured using a Voluson i ultrasound device (General Electric Healthcare Systems, Vienna, Austria) equipped with a 12-MHz, multi frequency, linear array probe. Serum concentrations of progesterone were determined from blood samples collected at each ultrasound examination. Images of the CL were captured and stored for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow area were captured and stored for analysis of luteal blood flow ratio. Longitudinal B-mode images of the uterine horns were stored for analysis of echotexture. Diagnosis of pregnancy was made at each ultrasound examination based on CL blood flow, CL size, and uterine echotexture. Pregnancy was confirmed by ultrasonography on d 30 after AI. The relationship between ultrasound measures and pregnancy outcome, as well as the accuracy of the pregnancy diagnosis made at each ultrasound examination was assessed. Progesterone concentrations and CL tissue area were greater in pregnant compared with nonpregnant cows on all days. The CL blood flow ratio was higher in pregnant compared with nonpregnant cows on d 20 and 21 after AI. Echotexture measures of the CL and uterus were not different between pregnant and nonpregnant cows on any day of examination. The best logistic regression model to predict pregnancy included scores for CL blood flow, CL size, and uterine echotexture on d 21 following AI. Accuracy of pregnancy diagnosis was highest on d 21, with sensitivity and specificity being 97.6 and 97.5%, respectively. Uterine echotexture scores were similar for pregnant and nonpregnant cows from d 18 to 20. On d 21, pregnant cows had higher uterine echotexture scores compared with nonpregnant cows. The logistic regression equation most likely to provide a correct pregnancy diagnosis in lactating dairy cows included the visual score for CL blood flow, CL size, and uterine echotexture on d 21 after AI. In support of this finding, the diagnostic accuracy for visual scores of CL blood flow, CL size, and uterine echotexture were also highest on d 21.

Paradoxical effect of supplementary progesterone between Day 3 and Day 7 on corpus luteum function and conceptus development in cattle.

The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRID® Delta; 1.55 g P4) treatment with administration of a prostaglandin F(2α) analogue (Enzaprost; CEVA Sante Animale) the day before PRID® Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRID® Delta from Day 3 to Day 7; (4) insertion of a PRID® Delta from Day 3 to Day 5; or (5) insertion of a PRID® Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n=10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-τ (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P<0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0±1.4 mm) or Day 3 to Day 7 (11.3±2.3 mm) increased conceptus length compared with placebo (2.1±1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P<0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0±18.8 mm) or Day 3 to Day 7 (143.6±20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3±17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P<0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r=0.58; P=0.011) and spent culture medium (r=0.68; P<0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

Physiological status alters immunological regulation of bovine follicle differentiation in dairy cattle.

Lactation in dairy cattle is associated with a multitude of endocrine, metabolic and immunological changes that not only influence animal health, but also affect fertility, and in particular ovarian function. We have previously generated a global transcriptomic profile of bovine follicular tissue using RNA sequencing. This study aimed to: identify key immune-related transcriptional changes that occur during follicle differentiation and luteinisation using ingenuity pathway analysis (IPA); and determine if a compromised model of stress (i.e. lactation) influences the temporal expression of these genes. Ovarian follicular tissue from Holstein-Friesian non-lactating heifers (n=17) and lactating cows (n=16) was compared at three stages of preovulatory follicle development: (A) the newly selected dominant follicle in the luteal phase (Selection); (B) the follicular phase before the LH surge (Differentiation), and (C) the preovulatory phase after the LH surge (Luteinisation). IPA revealed an over-representation of immune-related pathways in theca compared with granulosa cells during differentiation; these were related to leucocyte extravasation and chemotaxis. Conversely, luteinisation was characterised by over-representation of immune-related pathways in granulosa compared with theca cells; these were related to inflammation and innate immune response. Notably, comparison of follicles from heifers and lactating cows revealed a large number of differentially expressed genes associated with immune cell subpopulations and chemotaxis. In conclusion, identification of immune-related canonical pathways during follicle development supports the hypothesis that ovulation is an inflammatory event. This process is influenced by the physiological status of lactation and likely contributes to compromised peri-ovulatory follicle function by impairing the inflammatory process of ovulation.