Cyclic Heifers Research Articles

Characterizing emergence and divergence in the first follicular wave in a tropically adapted Bos taurus breed

Tropically adapted Bos taurus breeds are descended from the cattle which were brought from the Iberian Peninsula to Latin America by the colonizers and which adapted over time to local conditions. These breeds have become a genetic treasure that must be preserved. The objective of this study was to characterize ovarian follicular emergence and divergence in tropically adapted B. taurus cows and heifers. Cyclic heifers (n = 11) and nonlactating, multiparous cows (n = 11) were examined at 8-hour intervals using ultrasonography starting on the day following the final application of PGF2α, which was used to synchronize the estrous cycle, and ending 5 days after ovulation. Blood samples were collected immediately following the ultrasonographic examinations in order to evaluate FSH and estradiol-17β (E2) concentrations. The interval between ovulation and follicular wave emergence (11.6 ± 3.3 hours vs. 20.3 ± 2.5 hours, P < 0.05) and ovulation and follicular wave divergence (52.4 ± 5.2 hours vs. 71.8 ± 4.1 hours, P < 0.05) was shorter in the cows than in the heifers, respectively. Plasma FSH concentrations increased (P < 0.05) and serum E2 concentrations decreased earlier in cows than in heifers before ovulation. Following follicular wave emergence, no difference in follicular development was found between the cows and the heifers. Consequently, following follicular wave emergence, the data from both the cows and the heifers were combined and categorized by dominant follicle (DF) and second largest follicle (SF). The DF and SF were identified at the same time (P > 0.05). The mean number of small (≤4 mm, 7.2 ± 5.1) and medium (4 to ≤ 8 mm, 6.8 ± 3.5) follicles was greater than that of large follicles (≥8 mm, 0.6 ± 0.5) from ovulation until 5 days after ovulation. The DF diameter (8.1 ± 1 mm) did not differ (P = 0.09) from SF diameter (7.6 ± 0.9 mm) at the time of follicular divergence (around 45 hours after follicular emergence). The DF and SF growth rates were similar (P > 0.05) until follicular divergence, at which point the SF growth rate decreased, whereas the DF growth rate remained constant. Serum E2 concentrations did not change (P > 0.05) during the divergence period, whereas FSH concentrations decreased between 48 and 32 hours before follicular wave divergence. In conclusion, cows and heifers differed only in the interval from ovulation to follicular wave emergence and divergence, which was confirmed by the different patterns of FSH and E2 concentrations.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Spatial differences in gene expression in the bovine oviduct.

The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.

Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers.

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

Ovarian response and conception rate following oestrus synchronization using three protocols in Egyptian buffalo heifers.

The aim of this study was to monitor the ovarian response and conception rate following estrous synchronization using CIDR, Ovsynch and double prostaglandin F2α protocols in Egyptian buffalo heifers. A total of 80 cyclic buffalo heifers were divided into four equal groups: CIDR (intravaginal progesterone releasing device, EAZI-BREEDTM CIDR®), Ovsynch (GnRH, PGF2α, GnRH injections), PGF (double PGF2α doses) and control. Timed artificial insemination (TAI) was performed in all heifers. All animals were examined using ultrasound and blood samples were collected for measurement of progesterone. A new follicular wave occurred earlier in the Ovsynch and PGF groups than in the CIDR group (p<0.05). The mean diameter of the ovulatory follicle was smaller in the CIDR group than in the Ovsynch and PGF groups (p<0.05). The ovulation rate was 100% in the CIDR group, 75% in the Ovsynch group and 70% in the PGF group. In the control group a lower pregnancy rate (20%) was determined in than in the CIDR (35%), Ovsynch (40%) and PGF (35%) groups. Progesterone concentrations were numerically higher in pregnant heifers of the CIDR group but the difference was non-significantly compared to the Ovsynch, PGF and control groups (p>0.05). EAZI-BREEDTM CIDR®, Ovsynch-based TAI and PGF protocols were effective in synchronizing oestrus and resulted in nearly similar pregnancy rates in Egyptian buffalo heifers.

Effectiveness of Heat Detection Patches for Determining Cyclicity Status in Peripubertal Heifers

Response to most estrous synchronization programs is improved in cycling females, compared to pre-pubertal heifers or anestrus cows. Cycling status is often monitored for research purposes by measuring progesterone concentrations in serum. Producers may want to use information on cyclicity status when determining which estrous synchronization program to use or as a component of their heifer selection. Heat detection patches are routinely used to aid or reduce time needed for heat detection. The most data on their use is for a 3 to 5-day period after estrous synchronization. Little information is available on retention of patches for longer time periods or how they may compare with serum progesterone for determining cyclicity status. Therefore, the objective of the current study was to compare Standing Heat and Estrotect heat detection patches with serum concentrations of progesterone for identifying cycling (pubertal) heifers before the start of the breeding season.

73 PROTEOMIC ANALYSIS OF UTERINE LUMINAL FLUID ON DAY 7 OF PREGNANCY IN CATTLE

In vitro fertilization studies have demonstrated that contact with the female reproductive tract is not necessary in order for the embryo to reach the hatched blastocyst stage. Furthermore, the endometrial transcriptome does not differ between cyclic and pregnant cattle before maternal recognition around Days 15 to 16, suggesting that before that time the cow does not know she is pregnant. Nonetheless, embryo quality is significantly improved by exposure to reproductive tract secretions. Therefore, the aims of this study were to (1) characterise the protein content of uterine luminal fluid (ULF) on Day 7 of pregnancy and the oestrous cycle; (2) identify if differences in ULF composition exist between pregnant and cyclic heifers on Day 7; and (3) determine if blastocyst-derived proteins contribute to ULF composition. Following oestrus synchronization, heifers observed in standing oestrus were randomly assigned to either inseminated (AI: n = 12) or cyclic control (C: n = 6) groups. At slaughter, the uterine horn ipsilateral to the corpus luteum was flushed with 10 mL of PBS. In the AI group, the presence of an appropriately developed embryo was noted (n = 8), and the ULF of both confirmed pregnant and cyclic heifers was clarified by centrifugation at 1000 × g for 15 min at 4°C. Blastocyst-conditioned medium was produced by culturing in vitro-produced blastocysts (n = 300) for 24 h in synthetic oviduct fluid (SOF; groups of 50 blastocysts in 500 µL of SOF). After 24 h the conditioned medium (n = 6 pools), along with contemporaneous blanks (n = 6), was collected and snap frozen. Proteomic analysis of ULF as well as blastocyst-conditioned medium was carried out by nano-LC tandem mass spectrometry (nano-LC-MS/MS). A total of 661 proteins were identified in the ULF of heifers on Day 7 with &gt;95% confidence in at least 4 out of the 6 animals analysed per group. The most abundant proteins were myosin-9, cytoplasmic dynein 1 heavy chain 1-like, fatty acid synthase isoform X1, serum albumin, and aminopeptidase N. No differences were detected between pregnant and cyclic ULF. Of the proteins identified, the biological processes of gene expression (23.8%), small molecule metabolic process (22.6%), and cellular protein metabolic process (17.8%) were most overrepresented. Interestingly, 400 proteins were associated with extracellular exosome as well as extracellular space (87 proteins) and extracellular region (58). Analysis of blastocyst-conditioned medium identified 11 proteins that were not detected in the contemporaneous blanks, of which one (α-1-acid glycoprotein) was also detected in Day 7 ULF. In conclusion, no differences in the ULF composition between pregnant and cyclic heifers were observed on Day 7, consistent with studies on the endometrial transcriptome. A large proportion of the proteins detected are associated with the extracellular space region and may represent candidate proteins that can be added to embryo culture media to enhance early development. Research was funded by Science Foundation Ireland (13/IA/1983).

12 INITIAL GONADOTROPIN-RELEASING HORMONE TREATMENT INCREASED PREGNANCY PER TIMED INSEMINATION ONLY IN ACYCLIC BEEF HEIFERS SUBJECTED TO A 5-DAY CO-SYNCH PROTOCOL

We have shown that inclusion of initial gonadotropin-releasing hormone (GnRH) in a 5-day Co-synch protocol is unnecessary to achieve acceptable pregnancy rates in dairy heifers (2011 Theriogenology 76, 578). This study determined the efficacy of a modified (without initial GnRH) 5-day Co-synch protocol for timed AI (TAI) and the effect of interval from progesterone device removal to TAI on pregnancy per TAI (P/TAI) in acyclic and cyclic beef heifers. Heifers (n = 1062; 302 to 515 kg of body weight, and 13 to 15 months of age) at 2 locations were assigned to either a 5-day Co-synch protocol with (control) or without (modified) an initial injection of 100 μg of GnRH (Fertagyl; Merck Animal Health, Kenilworth, NJ, USA) at progesterone device (CIDR; Zoetis Animal Health, Florham Park, NJ) insertion (Day 0). All heifers received a single dose of 500 μg of cloprostenol (Estrumate; Merck Animal Health) at CIDR removal (Day 5) and were divided into 2 groups to receive GnRH and simultaneously TAI at either 66 or 72 h (Day 8) after CIDR removal. All heifers were inseminated by one technician with frozen-thawed semen from 1 of 4 commercially available sires. Ovarian ultrasonography was performed on Day 0 to determine cyclicity (presence of corpus luteum) and normalcy of the reproductive track, and on Day 35 (27 days after TAI) to determine pregnancy status. Data were analysed using the PROC GLIMMIX in SAS (SAS Institute Inc., Cary, NC, USA). The CIDR retention rate was 98%; P/TAI in heifers that lost the CIDR was 39%. Pregnancy per TAI [least squares means (n)] did not differ (P &gt; 0.05) among sires [48 (235), 51 (318), 52 (274), and 53% (235)], location [57 (330) v. 53% (732)], timing of TAI [52 (533) v. 50% (529) for 66 and 72 h], or treatment groups [48 (525) v. 53% (537) for control and modified groups]. A total of 226 (21%) heifers were acyclic; P/TAI was lower in acyclic heifers compared with that in those cyclic (P &lt; 0.01; 43 v. 59%). Administration of GnRH at CIDR insertion increased P/TAI in acyclic heifers (P &lt; 0.01; 50 v. 35% for control and modified groups) but did not affect P/TAI in those that were cyclic at initiation of protocol (P &gt; 0.05; 57 v. 62% for control and modified groups). In conclusion, timing of TAI did not affect fertility. However, administration of initial GnRH increased P/TAI by 15 percentiles in acyclic females, but as previously shown in Holstein heifers, initial GnRH was unnecessary to achieve acceptable P/TAI in cyclic beef heifers subjected to a 5-days Co-synch protocol. This study was supported by Alberta Agriculture and Forestry, Intervet Canada Corp., and collaborative beef producers.

108 FACTORS AFFECTING PREGNANCY RATES IN RECIPIENTS RECEIVING IN VITRO-PRODUCED EMBRYOS BY FIXED TIME EMBRYO TRANSFER

A retrospective analysis of factors that affect pregnancy rates from 4214 fresh in vitro-produced (IVP) embryos that were transferred at a fixed-time (FTET) in 20 different farms. Recipients were all cycling cows or heifers that were synchronized with 1 of 3 treatments: 1) treatments with progesterone (P4) devices and 2 mg of oestradiol benzoate (EB) on Day 0 (day of insertion) and 24 h after device removal (Day 8); 2) treatments with P4 devices and EB on Day 0, but with 0.5 mg of oestradiol cypionate (ECP) at device removal (Day 8); or 3) treatments with P4 devices and GnRH on Day 0 and a second GnRH 60 h after device removal (Day 5). Cows in all treatment groups also received 500 µg of cloprostenol (prostaglandin F2α) at the time of P4 device removal and 400 IU of eCG either at device removal or 3 days before device removal. All embryos were transferred 7 or 8 days after the expected time of oestrus (24 h after EB, 48 h after ECP or at the time of the second GNRH for each synchronization treatment, respectively). On the day of embryo transfer, recipients were examined by ultrasonography and those with corpus luteum &gt;14 mm in diameter received a fresh, IVP embryo in the uterine horn ipsilateral to the corpus luteum. Pregnancy rates were determined by ultrasonography 35 days after FTET. Data were analysed by logistic regression. Independent variables were classified into the following three categories. 1) Factors related to the recipient and the environment; there were no significant differences in pregnancy rates for corpus luteum diameter (≥14 and &lt;16 mm, ≥16 and &lt;18 mm, or ≥18 mm; P = 0.46), number of corpus luteum (1 or ≥2; P = 0.26), and category of recipient (cow or heifer; P = 0.21). However, there were significant effects of farm (P = 0.01) and body condition score (BCS; P = 0.01). Cows with BCS ≥4.5 (1 to 5 scale) resulted in lower pregnancy rates (4/20, 20.0%) than those with BCS 2 (74/225, 32.9%), 2.5 (502/1434, 35.0%), 3 (570/1467, 38.9%), 3.5 (193/532, 36.3%), and 4 (44/118, 37.3%). 2) Factors related to the synchronization treatment; there were no significant differences between recipients receiving eCG at device removal (84/209, 40.2%) or 3 days before device removal (874/2291, 38.1%; P = 0.35). However, recipients synchronized with P4 devices and ECP had higher (P = 0.01) pregnancy rates (232/483, 48.0%) than those treated with EB (679/1888, 36.0%) or gonadotropin-releasing hormone (47/129, 36.4%). 3) Factors related to the embryo transfer technique; day of the recipient’s oestrous cycle (P = 0.36), stage of embryo transferred (IETS stages 6 or 7; P = 0.62), and operator (P = 0.57) did not affect pregnancy rates. However transfers made in the anterior third of the uterine horn resulted in higher (649/1545, 42.0%) pregnancy rates than those in the mid-third (845/2511, 33.6%) or in the distal third (6/35, 17.1%; P = 0.01). It was concluded that factors related to the recipient and the environment (farm and BCS), the synchronization treatment (ECP), and the embryo transfer technique (site of deposition) affect pregnancy rates in recipients of embryos produced in vitro and transferred at a fixed time.

Plasma anti-Müllerian hormone as a predictive endocrine marker to select Bos taurus (Holstein) and Bos indicus (Nelore) calves for in vitro embryo production

This study evaluated the association between plasma anti-Müllerian hormone (AMH) concentrations and in vitro embryo production in Bos indicus (Nelore; experiment 1) and Bos taurus (Holstein; experiment 2) calves superstimulated or not with 140 mg of porcine follicle-stimulating hormone (pFSH; 4 decreasing doses twice daily). Oocytes were recovered from calves aged 2 to 4 mo after receiving gonadotropin stimulation (Nelore, n = 15; Holstein, n = 12) or not (Nelore, n = 15; Holstein, n = 12). Cycling heifers formed a positive control group (n = 15 for Nelore [aged 18–24 mo], n = 10 for Holstein [aged 14–16 mo]). All the calves underwent laparoscopic ovum pickup, and cycling heifers underwent a regular transvaginal ultrasound-guided ovum pickup for oocyte recovery. Immediately before oocyte retrieval, blood samples were taken for subsequent AMH determination (ng/mL). Regardless of the genetic group, calves that received pFSH (3.6 ± 1.1 in Nelore and 4.6 ± 1.2 in Holstein) or did not receive pFSH (3.2 ± 1.0 in Nelore and 2.5 ± 0.8 in Holstein) had greater plasma AMH concentrations (P = 0.01 in Nelore and P = 0.003 in Holstein) than cycling heifers (1.1 ± 0.2 in Nelore and 0.6 ± 0.07 in Holstein). AMH concentrations in calves with or without pFSH were similar in both genetic groups (3.6 ± 1.1 vs 3.2 ± 1.0 in Nelore; 4.6 ± 1.2 vs 2.5 ± 0.8 in Holstein). In calves, positive correlations were observed between plasma AMH concentrations and the numbers of follicles >2 mm (r = 0.86, P < 0.0001 in Nelore; r = 0.78, P < 0.0001 in Holstein), cumulus-oocyte complexes (COCs) retrieved (r = 0.91, P < 0.0001 in Nelore; r = 0.82, P < 0.0001 in Holstein), COCs cultured (r = 0.71, P < 0.0001 in Nelore; r = 0.79, P < 0.0001 in Holstein), and blastocysts produced (r = 0.62, P = 0.0003 in Nelore; r = 0.58, P = 0.009 in Holstein), and these results were independent of pFSH treatment. In conclusion, calves had greater plasma AMH concentrations than cycling heifers. In addition, treatment with pFSH did not influence AMH concentrations in calves, regardless of the genetic group. More importantly, plasma AMH concentrations were positively correlated with the antral follicle population and the number of COCs retrieved, COCs cultured, and blastocysts produced in B indicus and B taurus calves. Therefore, AMH is a promising tool for selecting oocyte donor calves to maximize results during in vitro embryo production.

'Conceptualizing' the Endometrium: Identification of Conceptus-Derived Proteins During Early Pregnancy in Cattle.

The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16-18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.

Formulation and testing of a non-steroidal aromatase inhibitor intravaginal device for the control of ovarian function in cattle

The study was designed to formulate intravaginal devices that provide biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days, and to determine their physiologic effects in cattle. Three compounds with estradiol inhibitory capability (letrozole, anastrozole and fenbendazole) were tested in vitro using bovine granulosa cell culture. Letrozole was found to be the most efficient and potent inhibitor. A wax-based vehicle was selected for further development of a letrozole intravaginal device based on its steady release rate. Cycling heifers were assigned randomly to be given an intravaginal device containing wax plus gel coat (n=4), wax formulation (n=4), no formulation (blank device, control, n=4). Intravaginal devices were inserted on Day 3 (Day 0=ovulation) and kept in place for 8 days. The addition of a letrozole-containing gel coating hastened the initial increase on plasma concentrations, while the letrozole-containing wax-based vehicle maintained prolonged delivery from the intravaginal device. The dominant follicle diameter profile was larger in heifers treated with the wax plus gel coat device (P<0.04), and the interwave interval was prolonged in heifers in the letrozole-treated groups compared to controls (P<0.001). Plasma estradiol concentrations were reduced significantly in the letrozole-treated groups. Plasma progesterone concentrations were lower in the wax letrozole-treated group (P<0.02). We concluded that wax base plus gel coat intravaginal devices are suitable for the development of a letrozole-based protocol for the synchronization of ovulation in cattle. It effectively reduced estradiol production resulting in prolonged dominant follicle growth and lifespan, without adversely affecting progesterone production.

280 EFFECTS OF HOT SEASON ON BOVINE OOCYTE QUALITY: HOW TO BYPASS THE POOR OOCYTE QUALITY DURING THIS SEASON?

Many authors attribute the decline of reproductive activity in summer to the heat stress, a multifactorial problem in which hyperthermia affects cellular function in various tissues of the female reproductive tract (Hansen et al. 2001; De Rensis et al. 2003). In particular, the combination of high temperatures and high humidity for a long period causes a reduced blood flow to uterus, oviducts, and ovaries, leading to a rise in the concentration of the degradation products of cellular activity. Therefore, the aim of this work was to elucidate the negative effect of the hot season on bovine oocyte quality and evaluate the influence of different factors on the acquisition of meiotic competence. In particular, meiotic competence of bovine oocytes recovered from animals housed at 44°28′00″ N, 11°26′00″ E during spring (March, 4–13°C) and summer (June, 16–27°C) was evaluated. Likewise, in summer the effect of an antioxidant, myo-inositol, the use of serum replacement (SR), and the use of oocytes recovered from cycling heifers (16–18 months) as compared to cows (&gt;24 months) were tested. A total of 1346 abattoir-derived oocytes, equally divided for different experimental groups (over 6 replicates), were in vitro matured in TCM 199 supplemented with EGF (25 ng mL–1), IGF1 (100 ng mL–1), ITS supplement, pFSH-LH (0.1 IU each), and 10% FBS. Myoinositol was added at a concentration of 0, 15, 30, and 50 mM, while 10% SR was used alternatively to FBS. At the end of maturation period (20–22 h), oocytes were denuded and stained with 10 μg mL–1 of Hoechst 33342 at room temperature in the dark. After 15 min they were mounted on glass slides for evaluation of nuclear status using a Nikon Eclipse E400 microscope equipped with fluorescence filters. Nuclear configurations were classified as (a) germinal vesicle (GV), (b) germinal vesicle breakdown (GVBD), (c) metaphase I (M-I), (d) metaphase II (M-II), and (e) degenerated (DEG). Data are expressed as mean ± s.e.m. and were analysed by ANOVA (IBM SPSS Statistics) considering significance at P &lt; 0.05. Oocyte quality of summer oocytes was significantly lower than spring counterparts as result of a higher rate of DEG (8.2 ± 0.6 v. 0.7 ± 0.6) and GV (5.4 ± 0.3 v. 0.4 ± 0.4, respectively; P &lt; 0.05). Myo-inositol supplementation in IVM medium did not significantly affect either oocyte quality or meiotic competence in the hot season, such as the use of SR. When the oocytes were collected from cycling heifers ovaries during summer, the recovery rate of COC/ovary was significantly higher as compared to cows (4.5 v. 2.0), and a lower rate of DEG (1.8 ± 0.2; 8.2 ± 0.6) and GVBD (0.9 ± 0.6; 6.1 ± 0.3) was found (P &lt; 0.05), even if the rate of GV (22.4 ± 0.1 v. 5.4 ± 0.3) was higher (P &lt; 0.05) compared with cow. In conclusion, the hot season negatively affects oocyte quality, myo-inositol does not affect nuclear maturation, and SR can be used alternatively to FBS. The lower age of oocyte donor positively influenced the number of recoverable oocyte and degeneration rate.

LH secretion around induced ovulation during early and late diestrus and its effect on the appearance of short estrous cycles in cyclic dairy heifers

The aim of this study was to investigate the effect of the stage of the estrus cycle (early vs. late diestrus) on the LH secretion after induced ovulation and on the incidence of short estrous cycles in dairy heifers. Cyclic heifers were presynchronized with dexcloprostenol and divided into Groups D7 (n = 6) and D14 (n = 6). On Day 7 (D7) or Day 14 (D14) after ovulation (Day 0), all animals were treated with dexcloprostenol followed by 0.1 mg of gonadorelin (GnRH) 24 hours later. Blood samples were taken daily for progesterone (P4) analysis and for LH analysis every 10 minutes for 3 hours on Days 1, 3, and 5 and every 30 minutes for 6 hours starting just before GnRH administration. In a control group, Group C (n = 7), estrus was synchronized with an intravaginal progesterone-releasing device (CIDR) inserted for 9 days. Blood for P4 analysis was taken daily for 16 days and for LH analysis every 30 minutes for 31 hours starting 30 hours after CIDR removal (unmanipulated LH peak) and every 10 minutes for 3 hours on Days 1, 3, and 5 after ovulation. In all groups, ovarian structures and ovulation were detected via daily transrectal ultrasound examination around first and second ovulation. In D7, all cycles (6/6) were shorter than normal (range 7–9 days). In D14, 2 of the 6 animals had a short cycle (both 7 days) whereas 4 had a cycle of normal length. All cycles were of normal length in C. Animals in D7 and D14 were divided according to their cycle length into short (SC) and normal (NC) cycle groups. The mean size of the ovulatory follicle during the 3 days before ovulation was significantly different between D7 and D14 and on 3 days and 1 day before ovulation between SC and NC. Secretion of LH during the 6 hours after GnRH administration did not differ between D7 and D14, or SC and NC. The mean basal LH secretion in D7 and D14 on Days 1, 3, and 5 was significantly different. No difference on those days was noted between SC and NC. In conclusion, when ovulation is induced with GnRH 24 hours after giving dexcloprostenol, short estrous cycles can occur during both early and late diestrus. The preovulatory LH surge did not differ between D7 and D14, or SC and NC. Also, basal LH secretion on Days 1, 3, and 5 was similar in SC and NC, and lower basal LH concentration coincided with higher P4 concentration.

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.

Anti-Mullerian hormone concentration and antral ovarian follicle population in Murrah heifers compared to Holstein and Gyr kept under the same management.

This study was performed to evaluate plasma concentrations of anti-Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d-cloprostenol administered 14 days apart. After the second d-cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high- or low-AMH concentration based on the average within each genetic group, high-AMH heifers had greater (p < 0.0001) AFP than low-AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.

Plasma anti-mullerian hormone: an endocrine marker for in vitro embryo production from Bos taurus and Bos indicus donors

The aim of this study was to evaluate the association between plasma anti-mullerian hormone (AMH) concentration and in vitro embryo production (IVP) from Bos taurus (Holstein) and Bos indicus (Nelore) donors. A total of 59 Holstein (15 prepubertal heifers aged 8–10 mo, 15 cyclic heifers aged 12–14 mo, 14 lactating cows, and 15 nonlactating cows) and 34 Nelore (12 prepubertal heifers aged 10–11 mo, 10 prepubertal heifers aged 21–23 mo, and 12 cyclic heifers aged 24–26 mo) females were enrolled. All females underwent an ovum pick-up (OPU), without previous synchronization of the follicular wave, and IVP procedure. Immediately before the OPU procedure, blood samples were collected for subsequent AMH determination. A positive correlation was observed between the plasma AMH and number of in vitro embryos produced from Holstein (r = 0.36, P < 0.001) and Nelore (r = 0.50, P = 0.003) donors. For additional analyses, donors within each genotype were classified into 1 of 2 AMH categories (low or high) according to the average AMH concentration for each genotype. The results revealed that females classified as having high AMH presented a greater number of visible aspirated follicles (Holstein: 20.9 ± 1.5 vs 13.6 ± 0.9, P < 0.0001; Nelore: 54.3 ± 6.1 vs 18.6 ± 2.1, P < 0.0001) and a greater number of recovered cumulus-oocyte complexes (Holstein: 17.3 ± 1.5 vs 9.0 ± 0.9, P < 0.0001; Nelore: 45.3 ± 6.4 vs 13.4 ± 1.7, P < 0.0001). However, there was no difference in the blastocyst production rate (Holstein: 20.6% ± 4.0% vs 19.8% ± 4.2%, P = 0.60; Nelore: 33.7% ± 6.5% vs 27.4% ± 5.5%, P = 0.41, high and low AMH, respectively). Moreover, donors classified as having high AMH yielded a greater number of embryos produced per OPU (Holstein: 3.0 ± 0.7; Nelore: 7.0 ± 1.7) compared with those classified as having low AMH (Holstein: 1.2 ± 0.3, P = 0.04; Nelore: 2.2 ± 0.5, P = 0.007). In conclusion, although the plasma AMH concentration did not alter the ability of the cumulus-oocyte complex to reach the blastocyst stage, the AMH concentration in plasma can be an accurate endocrine marker for the in vitro embryo yield from either B. taurus (Holstein) or B. indicus (Nelore) donors. Therefore, AMH is a promising tool to enhance the overall efficiency of OPU–IVP programs in the field as a selective criterion for high embryo producing donors.

Feeding excess crude protein to gestating and lactating beef heifers: Impact on parturition, milk composition, ovarian function, reproductive efficiency and pre-weaning progeny growth

Angus–Simmental beef heifers confirmed pregnant to a single sire (n=78) were used to determine the effects of feeding excess CP during late gestation and early lactation on parturition, milk composition, reproduction, and pre-weaning progeny growth. At 192d in gestation, heifers were allotted by BW within BCS to receive either a corn silage-based control diet balanced to meet or exceed NRC recommendations (2000) (CON), or a diet that considerably exceeded CP requirements consisting of corn stover and DDGS, where DDGS were fed as a primary energy source (HP). Diets were formulated to provide similar daily energy intake (2.09Mcal/d NEg). Dietary treatments were stopped at 118±4d postpartum (DPP) and cow/calf pairs were comingled. Calving parameters and pre-weaning calf performance were assessed. At 64±4 DPP, milk samples were collected to determine composition and fatty acid profile. At 32±4 DPP, a single follicular wave was mapped via ultrasonography in 12 anestrous heifers per treatment. Starting 34±4 DPP, blood samples were taken every 10d until synchronization to determine resumption of estrous cyclicity. Gestation was longer (P=0.02) and incidence of dystocia was greater (P=0.003) for HP than CON, respectively. Birth BW was greater (P<0.001) in HP than CON progeny. However, milk fat, total solids and energy corrected milk production were greater (P≤0.03) in CON than HP, respectively. In contrast, milk urea N, MUFA, PUFA, and CLA content was greater (P<0.01) in HP than CON, respectively. During the anestrous period, dominant and secondary follicle diameter was greater (P≤0.05) and follicle wavelength tended (P=0.08) to be greater in HP than CON, respectively. Proportion of cyclic heifers at estrous synchronization did not differ between treatments, but HP tended (P=0.10) to resume estrous activity at fewer DPP than CON. Timed-AI and breeding season pregnancy rates did not differ (P≥0.30) between the HP and CON treatment. Calf BW was greater throughout the pre-weaning period (P<0.001) for HP than CON progeny. Results indicate that feeding DDGS as a primary energy source to first-parity heifers during late gestation and early lactation altered ovarian function and resulted in increased pre-weaning growth performance of progeny.

Influence of Lysophosphatidic Acid on Nitric Oxide-Induced Luteolysis in Steroidogenic Luteal Cells in Cows1

Lysophosphatidic acid (LPA) together with its active G protein-coupled receptors are present in the corpus luteum (CL) of the cow. Under in vivo conditions, LPA stimulated P4 and PGE2 secretion during the luteal phase of the estrous cycle in heifers. Furthermore, LPA maintained P4 synthesis and actions in the bovine CL in vitro. However, the effect of this phospholipid on nitric oxide (NO)-induced functional and structural luteolysis has not been investigated. The aim of the present work was to determine the effects of LPA on 1) NO-induced functional luteolysis, 2) NO-dependent PG synthesis, and 3) NO-induced structural luteolysis in cultured steroidogenic luteal cells. We documented that LPA reversed the inhibitory effect of NONOate, an NO donor, on P4 synthesis and PGE2/PGF2alpha ratio in cultured steroidogenic luteal cells. Additionally, LPA inhibited NO-induced apoptosis in cultured steroidogenic luteal cells via abrogation of the NO-dependent stimulatory influence on proapoptotic TNFalpha/TNFR1 and Fas/FasL expression, Caspase 3 activity, and the Bax/Bcl2 ratio during luteal regression in the bovine CL. In conclusion, this study proves that in the presence of LPA, NO cannot induce luteolytic capacity acquisition, leading to functional and structural luteolysis of bovine luteal cells.

77 OVIDUCT - EMBRYO INTERACTIONS: TWO-WAY TRAFFIC OR A ONE-WAY STREET? TRANSCRIPTOMIC RESPONSE OF THE BOVINE OVIDUCT TO THE PRESENCE OF AN EMBRYO

Despite clear evidence of a two-way interaction between the developing conceptus and the uterine endometrium in early pregnancy, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. The aims were (1) to characterise the transcriptome of the bovine oviduct at the initiation of embryonic genome activation (EGA), (2) to examine the effect, if any, of the presence of an embryo on the oviduct transcriptome, and (3) to compare gene expression in the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. The oestrous cycles of cross-bred beef heifers were synchronized and those recorded in standing oestrus were randomly allocated to control group, nonbred (n = 7), or AI group (n = 11). All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut in half (ampulla and isthmus). Each portion was flushed with 500 μL of PBS to confirm the presence of an oocyte/embryo and was then opened and scraped longitudinally to obtain epithelial cells. Cells were snap-frozen in liquid nitrogen for microarray analysis. All recovered oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. The recovery rate was 72.7% (8/11) and 83.3% (5/6) for pregnant and cyclic animals, respectively. The stage of the recovered embryos was as follows: 4-cell stage (n = 1), 8-cell stage (n = 5), and 8–16 cell stage (n = 2), whereas in the cyclic group all recovered structures were unfertilized oocytes. The cells of the isthmus from ipsilateral and contralateral oviducts from 5 cyclic and 5 pregnant animals (8-cell embryos) and the ipsilateral ampulla cells from the pregnant animals were used for microarray analysis (Affymetrix Bovine ST array, Affymetrix, Santa Clara, CA, USA). Array data were analysed using BioConductor packages in R and custom CDF files downloaded from MBNI. Preprocessing of raw data was performed with RMA, and differential expression was assessed by linear modelling implemented in the limma package. Genes displaying P &lt; 0.05 after adjustment for multiple testing were considered differentially expressed. A total of 18 809 probe sets were assessed for differential expression. Comparison of pregnant and cyclic oviduct epithelium revealed no significantly altered genes. However, comparison of the isthmus and ampulla of the ipsilateral oviduct in pregnant animals revealed 4011 (P &lt; 0.05) and 2327 (P &lt; 0.01) differentially expressed genes. Some of the gene ontologies involved in biological processes included fatty acid metabolism, cell adhesion, cell morphogenesis, cellular developmental process, and reproduction. In conclusion, we have characterised the transcriptome of the bovine oviduct epithelium at the initiation of embryonic genome activation on Day 3 post-oestrus in pregnant and cyclic heifers. Although large differences in gene expression were observed between the isthmus and ampulla, data suggest that the presence of an 8-cell embryo had no effect on the transcriptome of the cells of the isthmus, although a local effect at the precise position of the embryo cannot be ruled out.