Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.     

Comparison of two methods for recovery of ovarian oocytes from slaughter cattle

When aspiration was used, 80 oocytes were recovered from 185 ovarian follicles, whereas 155 oocytes were recovered by rupturing 154 isolated follicles (P < 0.01).Two oocytes were found in each of three isolated follicles, three oocytes in one follicle and no oocytes in four follicles.Of the oocytes recovered by aspiration, 45% were morphologically normal, whereas 63.2% morphologically normal oocytes were recovered from isolated follicles (P < 0.01).     

Estimate of the population of preantral follicles in the ovaries of Bos taurus indicus and Bos taurus taurus cattle

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.     

In vitro culture and karyotyping of oocytes from 4 to 6 week old lambs

A preliminary investigation was made into the meiotic development of oocytes taken from the ovarian follicles of 4–6 week old ewe lambs. In total 1097 oocytes were recovered from 8 lambs of normal karyotype, 5 lambs heterozygous for the Massey 1 Robertsonian translocation and 4 homozygous lambs. After culture 208 oocytes (19%) had resumed meiosis although in only 45 (4.1%) could the behaviour of the chromosomes at metaphase stages I and II be satisfactorily studied. This rather poor recovery was believed to be due to the stage of ovarian development coupled with technical deficiencies.The limited numbers prevent strict conclusions from being drawn but the present findings demonstrated a rather similar chiasma frequency at diakinesis for the three karyotypes (44 ± 3) and hinted that non-disjunction occurs at a higher frequency in the heterozygous female than those with normal or homozygous karyotypes.     

Factors affecting number of surface ovarian follicles and oocytes yield and quality in Egyptian buffaloes

Three experiments were conducted to evaluate factors affecting number of surface ovarian follicles and oocytes yield and quality in buffalo. In Experiment 1, ovaries (n = 126) were collected in pairs from slaughtered anoestrus, early pregnant and cyclic buffaloes. Ovarian follicles (1-3, 4-9 and > or = 10 mm diameter) were counted, aspirated and oocytes were recovered and evaluated. In Experiment 2, ovaries were divided into 2 groups. Group 1, ovaries bearing a CL (n = 74) and Group 2 non-bearing CL (n = 74), ovarian follicles (2-8 mm) were counted, aspirated and oocytes evaluated. In Experiment 3, oocytes were recovered using aspiration or slicing methods. In all experiments, oocytes were classified into good, fair, poor and denuded. Results showed that the development of small and total ovarian follicles are continuous and independent in early pregnant or cyclic buffalo cows, however, it significantly decreased (P < 0.01) in the ovaries of anoestrus buffaloes. Number of medium and large size follicles was significantly increased (P < 0.01) in cyclic buffaloes on Days 10-16 and 17-22 of oestrous cycle, while large follicles was significantly decreased (P < 0.01) in the ovaries of pregnant buffaloes. A significantly higher (P < 0.01) percentage of poor and denuded oocytes were recovered from ovaries of anoestrus and pregnant buffalo. While, the highest (P < 0.01) percentage of good quality oocytes were recovered from ovaries of cyclic buffaloes on Days 1-3 and 10-16 of oestrous cycle, eliciting that the stage of oestrous cycle is affecting the quality of buffalo oocytes. In addition, the presence of a CL stimulates the development of a significantly higher (P < 0.01) number ovarian follicles which produced a significantly higher (P < 0.05) number of good quality oocytes. Slicing of buffalo ovaries produced a significantly higher number of fair, poor and denuded oocytes. In conclusion, number of ovarian follicles and yield and quality of oocytes were affected by the reproductive status, stage of the oestrous cycle, presence of a CL and the method of oocytes retrieval.     

Effects of active immunization of prepubertal heifers against prostaglandin F2α, on the onset of puberty and subsequent ovarian function

The objective of these two experiments was to determine the effect of immunization of prepubertal heifers against prostaglandin F_2α conjugated to human serum albumin (PGF-HSA) on the onset of puberty and subsequent ovarian function. Heifers that were 2–3 months of age (n=34; mean ± standard error of the mean (SEM) bodyweight 75 ± 1.4 kg) were given a primary immunization (Day 0) and a booster 183 days later using one of two adjuvants; non-ulcerative Freund's adjuvant (NUFA) and diethylaminoethyl-dextran (DEAF-dextran) and one of two doses of PGF-HSA as immunogen (1 and 10 mg), with six to seven heifers per treatment; control heifers (n=7) were untreated. The experiment lasted up to 562 days. Plasma antibody titres every 2–3 weeks and progesterone concentrations two to three times weekly were determined. Oestrous behaviour was observed twice daily. In addition, the follicular status of ovaries of cyclic (n=4), prepubertal (n=6) or prolonged luteal phase ( > 200 days, induced by immunization against PGF) heifers (n=5) were monitored daily for 23 consecutive days by ultrasound examination. Antibody titres were higher (P < 0.05) immediately following booster immunization in heifers immunized with DEAF-dextran than with NUFA. High titres were maintained for longer (P < 0.05) in heifers immunized with NUFA than with DEAF-dextran. The mean number of oestrous periods was decreased (P < 0.05) in PGF-immunized heifers in comparison with control heifers following booster immunization (3.1, 0.5, 0.9, 1.5, 1.1 in control heifers and in heifers immunized with NUFA-1 mg, NUFA-10 mg, DEAE-dextran-1 mg and DEAE-dextran-10 mg, respectively. Puberty was delayed (P < 0.05), relative to control heifers, by 185 and 122 days (pooled standard error of the difference, 89) in heifers immunized with NUFA-1 mg and DEAE-dextran-10 mg, respectively. After first ovulation at puberty, persistent corpora lutea (CL) formed in two of six heifers immunized with NUFA-1 mg, five of seven immunized with NUFA-10 mg, two of seven immunized with DEAE-dextran-1 mg and one of seven immunized with DEAE-dextran-10 mg. The duration of persistence was 103 ± 19, 301 ± 33, 124 ± 23 and 100 days, respectively. The mean (±SEM) number of dominant follicles was not different (P > 0.05) for the three groups of heifers that were examined by ultrasound (3.7 ± 0.2, 3.8 ± 0.3 and 3.2 ± 0.3 for cyclic, prepubertal and persistent CL, respectively); neither was their rate of growth (1.8 ± 0.2, 1.5 ± 0.1 and 1.5±0.2 mm day⁻¹, respectively), rate of atresia (1.3 ± 0.1, 1.6 ± 0.1 and 1.8 ± 0.2 mm day⁻¹, respectively) or the maximum size of dominant follicles (11.2 ± 0.4, 9.6 ± 0.3 and 12.4 ± 1.8 mm, respectively). In summary, immunization of prepubertal heifers against PGF delayed the onset of puberty and reduced subsequent oestrous behaviour due to the formation of persistent CL following first ovulation and there was a similar growth pattern of dominant follicles before first ovulation in prepubertal heifers, cyclic heifers and heifers which have long-term persistent CL.     

Comparison of oocyte developmental competence and follicular steroid content of nulliparous heifers and cows at different stages of lactation

Reduced reproductive performance and lower conception rates of lactating cows are closely associated with genetic progress for high milk production. In contrast, the fertility of nulliparous Holstein heifers has remained fairly stable over the years and appears to be markedly higher than that of mature lactating cows. Possible differences in oocyte quality and follicular steroid levels, which could be associated with the low fertility of high-lactating cows, were examined in 13-month-old heifers, cows around the time of first AI (60-95 d post-partum, yielding 49+/-2.4 kg/d) and cows at mid-lactation (120-225 d post-partum, yielding 37+/-2.1 kg/d). Estrus was synchronized by two doses of PGF2alpha and follicles (5-8 mm) were aspirated on days 4, 8, 11 and 15 of the cycle by an ultrasound-guided procedure. Oocytes were morphologically examined, matured in vitro, chemically activated and cultured for 8d. Cleavage rate and the proportion of developing parthenogenetic blastocysts were determined on days 3 and 8 post-activation, respectively. On day 17, heifers and cows received additional PGF2alpha and follicular fluids from preovulatory follicles were collected on day 19 perior to the expected estrus. Follicular-fluid volumes were similar in cows and heifers, as were estradiol, progesterone and androstenedione concentrations in the follicular fluid. Percentages of high-grade oocytes, proportions of cleaved oocytes and developed blastocysts did not differ between the groups. Results suggest that the fertility gap between nulliparous heifers and high-lactating cows is not directly related to steroid content in the preovulatory follicular fluid or oocyte developmental competence.     

Follicle populations and gene expression profiles of Nelore and Angus heifers with low and high ovarian follicle counts

The number of antral follicles counted (AFC) by ultrasound is associated with fertility in cattle. Cows with higher follicle count (HFC) have higher performance in reproductive‐assisted technologies than cows with lower follicle count (LFC). In this study, we aimed to define the preantral follicle count by histology and to identify differentially expressed genes (DEGs) using a microarray in Nelore and Angus heifers with HFC and LFC. The ovaries of each animal were scanned with an ultrasound device 12 to 24 hr after estrus. The groups were formed based on the average number of total follicles (≥3 mm) counted in each breed consistently ± the standard deviation. For the histological analysis, preantral follicles were counted and classified under a stereo microscope, and follicle density was determined. Microarray analysis was performed on pools of three follicles dissected from the ovaries of 15 Nelore (6 HFC and 9 LFC) and 17 Angus heifers (9 HFC and 8 LFC). Angus heifers have increased total and primordial follicle density. Nelore heifers have increased antral follicle count. Different patterns of gene expression regulate follicle recruitment and development in Angus and Nelore heifers and may be associated with the different follicle densities observed in Angus versus Nelore heifers. Furthermore, HFC heifers presented increased expression of genes associated with cellular development and metabolism.     

Differential abundance of IGF1, bile acids,and the genes involved in their signaling in the dominant follicle microenvironment of lactating cows and nulliparous heifers

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30–50 days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.     

Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers

A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)-stimulated (superovulated) and unstimulated heifers. Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol-17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy. The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid-mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH-stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley-Liss, Inc.     

Two different schemes of twice-weekly ovum pick-up in dairy heifers: effect on oocyte recovery and ovarian function

The aim of the present study was to compare two different schemes of twice-weekly ovum pick-up (OPU), continuous (C) and discontinuous (DC), with special emphasis on differences in oocyte yield and quality, estrous cyclicity, ovarian dynamics, and progesterone production. Subsequent to characterization of their normal estrous cycles (pre-OPU period), eight dairy heifers were subjected to 4 months of twice-weekly OPU under two different schemes: the DC (OPU restricted to Days 0-12 of the cycle) and the C schemes. Effects of the two different schemes on oocyte yield, quality, and in vitro competence, together with effects on ovarian dynamics and progesterone production, were monitored. The mean numbers of punctured follicles and recovered oocytes per session were slightly higher (not significant (n.s.)) using the DC scheme, but in total, similar numbers of oocytes were obtained. The quality of the oocytes as well as cleavage rate after in vitro fertilization of the oocytes did not differ between the two OPU schemes. There was no influence of a corpus luteum (CL) producing progesterone on the oocyte yield and quality, whereas the presence of dominant follicles appeared to decrease the number of recovered ooctyes. During the pre-OPU period, all heifers showed normal cyclicity. In the DC scheme, the heifers showed regular and normal cyclic activity throughout the puncture period, with one to two complete follicular waves during the interval from the last OPU to the next estrus. In the C scheme, the heifers occasionally revealed cyclicities with irregular interestrous intervals and weaker signs of estrus. No complete follicular waves were seen during the OPU period in this scheme. The CL developed from the ovulation of the preovulatory follicles in the DC scheme showed similar characteristics to the CLs of the pre-OPU period; however, the CL-like structures from the puncture of follicles, in both the DC and the C schemes, revealed a shorter life span and inferior competence in producing progesterone (P<0.05). The present results indicate that the DC OPU scheme, which allows animals to go into natural ovulation prior to the first OPU, does not affect their ovarian function, whereas the C OPU scheme does. Our study further demonstrates that an equal number of oocytes can be obtained with both schemes, but that fewer OPUs are needed when the DC scheme is applied.     

Development during single IVP of bovine oocytes from dissected follicles: Interactive effects of estrous cycle stage,follicle size and atresia

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles ≥3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P < 0.001) over all follicle size categories (3–5 mm, 6–8 mm, 9–12 mm and ≥13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 ± 0.86 over all size categories), particularly those of medium size (25.55 ± 2.2 for 6–8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 ± 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia. Mol. Reprod. Dev. 53:451–458, 1999. © 1999 Wiley‐Liss, Inc.     

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.     

Ovarian follicular growth suppression by long-term treatment with a GnRH agonist and impact on small follicle number,oocyte yield,and in vitro embryo production in Zebu beef cows

The aim of the present study was to evaluate small follicle number, oocyte yield, and in vitro embryo production (IVEP) in Zebu beef cows treated long term with a GnRH agonist to suppress ovarian follicular growth. Nelore (Bos indicus) cows (n = 20) showing regular estrous cycles were randomly assigned to one of two groups: control (n = 10, placebo ear implant without a GnRH agonist); GnRH agonist (n = 10, GnRH agonist ear implant containing 9.4-mg deslorelin). All cows underwent an ovum pick-up (OPU) session 14 days (Day 14) before the start of treatments (Day 0) followed by seven OPU–IVEP procedures at 30-day intervals (Days 0, 30, 60, 90, 120, 150, and 180). Semen from a single batch of a previously tested bull was used for all the IVEP. Cows treated with agonist reported a decrease over time in the proportion of animals with a (CL; P ≤ 0.05) and large follicles (>10 mm, P ≤ 0.05). These cows had a lesser number of medium + large follicles (>5 mm; 1.74 ± 0.5 vs. 4.13 ± 0.5; P ≤ 0.05), greater number of small follicles (2–5 mm; 44.3 ± 2.8 vs. 30.8 ± 1.8; P ≤ 0.05), greater yield of cumulus-oocyte complexes (COCs; 21.0 ± 2.3 vs. 15.6 ± 1.9; P ≤ 0.05), greater proportion of COCs cultured (79.2 vs. 73.9%; P ≤ 0.05), COCs cleaved (10.6 ± 1.5 vs. 6.8 ± 1.1, P ≤ 0.05), and cleaved rate (52.8 vs. 44.3%; P ≤ 0.05) compared with control cows. The number (3.4 ± 0.7 vs. 3.0 ± 0.6; P > 0.05) and proportion (16.5 vs. 19.1%; P > 0.05) of blastocysts produced were similar between agonist and control cows, respectively. The study has shown that Zebu beef cows treated long term with a GnRH agonist had follicular growth restricted to small follicles. This did not compromise the ability of oocytes to undergo IVF and embryonic development.     

Estrus characterization in superovulated cattle

Based on frequent examinations per rectum and on external signs of estrus, the intensity of estrus was characterized in 107 superovulated dairy cows and heifers. Three intensity groups were defined: strong (n=57), moderate (n=34), and weak (n=16). Frequent blood samples were obtained for analyses of estradiol-17β and LH. The ovaries were exposed through surgery at various intervals after the time of prostaglandin administration, and ovarian findings (follicles, premature ovulations) were described. A total of 527 oocytes was recovered from 998 follicles aspirated, and, of these, 420 were processed for chromosomal evaluation of their nuclear maturational stage.

The weak estrus intensity group differed from the strong and moderate groups in several ways. Cows and heifers in the weak intensity group had more frequent deviations in plasma hormonal patterns, a higher proportion of animals with only a few follicles, and more animals exhibiting premature ovulations. The mean oocyte recovery rate was lower in the weak group as compared with that of the other 2 groups, since more oocytes could not be accounted for in this group during the evaluation process, indicating a higher degree of oocyte fragility. Furthermore, a larger number of oocytes in the weak group displayed no identifiable chromosomes, than in the remaining 2 groups. However, apparently normal oocytes were also found in the weak group.

It is concluded that accurate characterization of estrus in superovulated cattle can be a valuable tool for identifying donors with a high risk for inadequate oocyte maturation, which in turn could lead to inferior embryo yield.     

Use of GnRH agonist implants for long-term suppression of fertility in extensively managed heifers and cows

The ability of gonadotrophin releasing hormone (GnRH) agonist implants to suppress ovarian activity and prevent pregnancies, long-term, was examined in heifers and cows maintained under extensive management. At three cattle stations, heifers (2-year-old) and older cows (3- to 16-year-old) were assigned to a control group that received no treatment, or were treated with high-dose (12 mg, Station A) or low-dose (8 mg, Station B and Station C) GnRH agonist implants. The respective numbers of control and GnRH agonist-treated animals (heifers + cows) at each station were: Station A, 20 and 99; Station B, 19 and 89; Station C, 20 and 76. Animals were maintained with 4% bulls and monitored for pregnancy at 2-monthly intervals for approximately 12 months. Pregnancy rates for control heifers and control cows ranged from 60-90% and 80-100%, respectively, depending on the study site. The respective number of animals (heifers + cows) treated with GnRH agonist that conceived, and days to first conception, were: Station A, 9 (9%) and 336 +/- 3 days; Station B, 8 (10%) and 244 +/- 13 days; Station C, 20 (26%) and 231 +/- 3 days. Treatment with high-dose GnRH agonist prevented pregnancies for longer (approximately 300 days) than treatment with low-dose GnRH agonist (approximately 200 days). In the majority of heifers and cows treated with GnRH agonist, ovarian follicular growth was restricted to early antral follicles (2-4mm). The findings indicate that GnRH agonist implants have considerable potential as a practical technology to suppress ovarian activity and control reproduction in female cattle maintained in extensive rangelands environments. The technology also has broader applications in diverse cattle production systems.     

Comparison of ovulation, fertilization and embryonic survival in low-fertility beef cows compared to fertile females

The objective was to determine physiological causes of low fertility in beef cows. Fertility was compared between low-fertility cows (34 British cows and 64 Brahman crossbred cows; cows that did not get pregnant when mated to fertile bulls in one or two previous breeding seasons); fertile cows (16 Brahman crossbreds; cows having a calf in several of the preceding breeding seasons), and virgin heifers (45 Brahman crossbreds, 2 yr of age). Females were mated to fertile bulls and killed 3 or 34 d after breeding to obtain reproductive tracts. There were no significant differences among groups in rates of ovulation or fertilization. Overall, 14% of females failed to ovulate and 24% that ovulated failed to undergo fertilization. The proportion of cows that were not detected in estrus before Day 34 of pregnancy was lower (P < 0.01) for low-fertility British cows (5 of 16 cows, 31%) than for other groups, including low-fertility Brahman crossbred cows (23/32, 72%), fertile cows (8/9, 89%), and heifers (21/24, 88%). All cows that did not return to estrus by Day 34 had an identifiable conceptus. The proportion of conceptuses recovered at Day 34 that were classified as normal (weight and length) was lower (P < 0.05) for cows with low fertility (British: 2/5, 40%; Brahman crossbred: 9/23, 39%) than for fertile cows (8/8, 100%) or heifers (18/21; 86%). Similarly, the proportion of cows in which a normal embryo was recovered (cows with normal embryos/number of cows mated) was lower (P < 0.001) for low-fertility British cows (2/16, 13%) and low-fertility Brahman crossbred cows (9/32, 28%) than for fertile cows (8/9, 89%) and heifers (18/24, 75%). In conclusion, cows that were infertile in previous breeding seasons did not experience reduced ovulation or fertilization rates, but had greater embryonic mortality. These data highlighted the importance of ovulation and fertilization failure and embryonic mortality as important determinants of pregnancy success. Moreover, increased embryonic loss after Day 34 contributed to infertility in low-fertility cows.     

Potential use of ovum pick-up for embryo production and breeding in cattle

The efficacy of transvaginal ultrasound-guided puncturing of ovarian follicles for collecting immature oocytes in cattle was studied. Three experiments were conducted to examine the effects of puncturing on follicle recruitment and on the number of oocytes collected. Puncture sessions were executed twice weekly at regular intervals of 3 and 4 d respectively. The oocytes were matured, fertilized and allowed to develop in vitro and the number of transferable embryos was recorded. The health of the cows was checked daily. In Experiment 1, dairy cows (n=10) were punctured over a period of 5 mo, and the collected oocytes were fertilized with the semen of 1 bull. In Experiment 2, oocytes were collected from one 12 year old high pedigree dairy cow and an one month pregnant cow were punctured. The oocytes of the old cow were fertilized with semen of 8 different bulls. In Experiment 3, beef cows (n=6) were punctured over a 2 mo period and the semen of 2 different bulls of the same breed was used to fertilize the oocytes from 3 of these cows. In Experiment 1, 14.5 +/- 0.4 (mean +/- SEM) follicles were punctured per session, and 8.0 +/- 0.3 (mean +/- SEM) oocytes were recovered. A mean of 16% of the oocytes developed into transferable embryos with a pregnancy rate of 40%. The results did not differ between the months of the experiments, indicating that the transvaginal puncturing method can be used successfully over a 5 mo period. No detrimental effects were observed after clinical and post mortem examinations, nor did breed, age or reproductive status appear to affect the results. However, large differences were observed between individual cows and between cow/bull combinations.     

The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts

The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons—summer (S: January to March) and winter (W: June to August)—and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus–oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding estrous cycle.