Developmental competence of oocytes from prepubertal Bos indicus crossbred cattle

The aim of the present study was to evaluate the effect of age on embryogenic competence of oocytes recovered from Bos indicus crossbred calves and heifers. Cumulus-oocyte complexes (COCs) were collected from 4- to 7-month-old calves (experiment 1) and from 9- to 14-month-old heifers (experiment 2) during processing at an abattoir. In both experiments cow COCs were used as control. COCs were in vitro matured and fertilized, and the presumptive zygotes co-cultured with cumulus cells until 224 h post insemination (hpi). In experiment 1, the development rate during the first 68-72 hpi was similar (P > 0.05) between embryos derived from calves and cows. Fewer embryos from calves developed to the blastocyst stage, resulting in a lesser blastocyst production as well as lesser hatching rate (P < 0.05). The embryo development after blastocyst stage was, nevertheless, similar (P > 0.05) between blastocysts derived from calves and cows, suggesting that the development after blastocoele formation is not compromised in embryos derived from calves. In experiment 2, there were no differences (P > 0.05) on cleavage, blastocyst and hatching rates between embryos derived from prepubertal heifers and cows. The rate of blastocyst development until hatching was also similar (P > 0.05). These results indicate that oocytes from 9- to 14-month-old B. indicus crossbred heifers have the same developmental competence as oocytes derived from cows, while ocytes derived from 4- to 7-month-old B. indicus crossbred calves are less competent in developing to the blastocyst stage in vitro. It suggests that oocyte competence in B. indicus crossbred cattle is achieved around 9-14 months of age.     

Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.     

Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts

The main objective was to test and validate a fluorescence approach to quantify lipid content of individual bovine oocytes and blastocysts. For Experiment 1, denuded oocytes were evaluated, as well as in vitro-produced blastocysts in a factorial design: cows versus feedlot heifers; three additives during Days 2.5-7.5 of culture (Control; 10% FCS; 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH); and two blastocyst stages (early versus expanded). All blastocysts were graded subjectively for darkness (1 = clear … 4 = dark). In Experiment 2, denuded oocytes were used to measure lipid content in a factorial design of: cows versus heifers and four subjective darkness grades (1 = clear … 4 = dark). To quantify lipids, oocytes and 7.5 d blastocysts were fixed and then stained with 1 μg/mL Nile Red dye in mPBS overnight. A digital photograph of the equatorial part of the oocyte and embryo was taken at 200×, and fluorescence intensity (Arbitrary Fluorescence Units, AFU) was measured with Image Pro software. Reverse images of the same photographs were used to count numbers of cytoplasmic lipid droplets of various sizes (LC). The linear regression equation of LC with AFU in oocytes had an r² = 0.84, and for blastocysts r² = 0.91. The LC and AFU also had similar coefficients of variation from the ANOVA for blastocysts (38 vs 44%, respectively). Treatment differences were of similar magnitude with both procedures: lipid content in oocytes and blastocysts from heifers and cows was similar (P > 0.1); PES reduced lipid accumulation, and FCS increased it relative to the Control for AFU (18.6 vs 46.6 vs 36.9 units, respectively), and LC (1763 vs 4081 vs 3310, respectively; all, P < 0.01). Early blastocysts resulted in more lipid accumulation per unit area than expanded ones based on AFU (41.5 vs 26.6) and LC (3519 vs 2583; both P <0.01). There was a strong relationship (P < 0.01) between subjective oocyte and blastocyst darkness and lipid content. The less labor intensive fluorescence staining was a reliable technique for quantifying lipid droplets in oocytes and blastocysts.     

The developmental competence of bovine nuclear transfer embryos derived from cow versus heifer cytoplasts

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos. In order for nuclear transfer to be successful, a differentiated donor cell must be reprogrammed and restored to a totipotent state. This reprogramming is probably accomplished by factors within the oocyte cytoplasm. This study indicates that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared to heifer oocytes based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages. Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes and resulted in higher pregnancy retention at 90 and 180 days and a greater number of term deliveries. Following delivery more calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.     

In vitro fertilization of ova from cows experimentally infected with a non-cytopathic strain of bovine viral diarrhea virus

Bovine viral diarrhea virus infection was induced in 16 heifers by inoculation of a noncytopathic strain of bovine viral diarrhea virus (BVDV). Six BVDV-free heifers served as controls. On Day 8 after inoculation, cumulus—oocyte complexes were collected from ovaries of animals at the second peak of fever preceded by leukopenia. The oocytes were then matured and fertilized in vitro. There was no significant difference (48% vs. 54% P>0.05) in the percentage of cleaved oocytes between infected and non-infected animals. However, the proportion of embryos that developed to the blastocyst stage was significantly higher for the control group than for BVDV group (29% vs. 14%) (P<0.01). All follicular fluids and cumulus—oocyte complexes collected from infected animals tested positive for the presence of the virus, but embryos produced by in vitro fertilization 7 days after in vitro co-culture tested negative.     

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.     

Studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle

Two experiments (Experiment I, n=12 Holstein-Friesian heifers; Experiment II, n=8 Jersey cows) were conducted to investigate the pathogenesis of bovine pestivirus-induced ovarian dysfunction in cattle. In both experiments the cattle were superovulated with twice daily injections of a porcine pituitary extract preparation of follicle stimulating hormone (FSH-P), for 4 days commencing on Day 10+/-2 after a presynchronised oestrus. The heifers received a total dose of 30 mg and the cows 32 mg of FSH-P. Prostaglandin F(2alpha) (PGF(2alpha)) was administered 48 h after commencement of superovulation and all cattle were artificially inseminated (AI) between 48 and 66h after PGF(2alpha) treatment. In both experiments bovine pestivirus seronegative cattle (Experiment I, n=6; Experiment II, n=4) were inoculated intranasally with an Australian strain of non-cytopathogenic bovine pestivirus (bovine viral diarrhoea virus Type 1) 9 days prior to AI. Bovine pestivirus infection was confirmed by seroconversion and/or virus isolation in all of the inoculated cattle, consistent with a viremia occurring approximately between Day 5 prior to AI and the day of AI. Ovarian function was monitored in both experiments by daily transrectal ultrasonography and strategic blood sampling to determine progesterone, oestradiol-17beta, luteinising hormone (LH) and cortisol profiles. Non-surgical ova/embryo recovery was performed on Day 7 after AI. In Experiment II half the cattle were slaughtered on Day 2 and the remainder on Day 8 after AI, and the ovaries submitted for gross and histopathological examination including immunohistochemistry to demonstrate the presence of bovine pestivirus antigen. In both studies, comparisons were made between infected and confirmed uninfected (control) animals. Overall the bovine pestivirus infected cattle had significantly lower (P<0.05) ova/embryo recovery rates compared to the control cattle. There was evidence of either an absence (partial or complete) of a preovulatory LH surge or delay in timing of the LH peak in the majority (90%) of infected heifers and cows, and histologically, there was evidence of non-suppurative oophoritis with necrosis of granulosa cells and the oocyte in follicles from the infected cows. By contrast only 20% of the control heifers and cows had evidence of absence of a pre-ovulatory LH surge. These experiments collectively demonstrate that bovine pestivirus infection during the period of final growth of preovulatory follicles may result in varying degrees of necrosis of the granulosa cells with subsequent negative effects on oestradiol-17beta secretion which in turn negatively affects the magnitude and/or timing of the preovulatory LH surge.     

Evaluation of steroid microspheres for control of estrus in cows and induction of puberty in heifers

Two trials were designed to test whether a single treatment with a microsphere formulation of progesterone (P) could simulate the luteal phase of the estrous cycle and lead to estrus and subsequent luteal development. The first experiment was to characterize the pattern of serum P concentrations and estrus in cows treated with a microsphere formulation (P + E) that contained 625 mg P and 50 mg estradiol (E). Four cows with palpable corpora lutea were treated with 25 mg prostaglandin F2 m. Each cow was given P + E (i.m.) 12 h later. Tail vein blood samples were taken on Days 1 and 2 following P + E treatment and then three times weekly for 24 days. Serum P increased from 0.8 +/- 0.1 ng/ml at P + E treatment to 4.7 +/- 0.6 ng/ml on Day 1, declined gradually to 4.1 +/- 0.3 ng/ml on Day 7 and then declined more rapidly to 0.6 +/- 0.1 ng/ml on Day 13. Treated cows showed estrus 16.25 +/- 0.7 days after P + E treatment. Thereafter, serum P increased beginning on Day 20 after P + E treatment, as expected following estrus. In Experiment 2, Angus and Simmental heifers (10.5-11.5 months of age) were administered i.m. either the vehicle (controls), E (50 mg), P (625 mg) or P + E (n = 13 per group). While treatment with E resulted in behavioral estrus (1-2 days after treatment) in each treated heifer, it did not (P > 0.5) initiate estrous cycles as indicated by subsequent increased serum P. In contrast, the P and P + E treatments increased (P < 0.05) the proportion (11/13) of heifers that showed estrus by 21 days after treatment followed by elevated serum P. We conclude that the microsphere formulation of P simulated the pattern of serum P concentrations during the luteal phase of the estrous cycle and initiated estrous cycles in peripubertal heifers with or without E.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

The use of progestins in regimens for fixed-time artificial insemination in beef cattle

Four experiments were conducted to investigate modifications to gonadotropin releasing hormone (GnRH)-based fixed-time Al protocols in beef cattle. In Experiment 1, the effect of reducing the interval from GnRH treatment to prostaglandin (PGF) was examined. Lactating beef cows (n = 111) were given 100 mg gonadorelin (GnRH) on Day 0 (start of treatment) and either 500 microg cloprostenol (PGF) on Day 6 with Al and 100 microg GnRH 60 h later, or PGF on Day 7 with Al and GnRH 48 h later (6- or 7-day Co-Synch regimens). Pregnancy rates were 32/61 (53.3%) versus 26/50 (52.0%), respectively (P = 0.96). In Experiment 2. cattle (n = 196) were synchronized with a 7-day Co-Synch regimen and received either no further treatment or a CIDR-B device (Days 0-7). Pregnancy rates were 32/71 (45.1%) versus 33/77 (42.9%) in cows (P < 0.8), and 9/23 (39.1 %) versus 17/25 (68.0%) in heifers (P < 0.05). In Experiment 3, 49 beef heifers were randomly assigned to receive 12.5 mg pLH on Day 0, PGF on Day 7 and 12.5 mg of pLH on Day 9 with Al 12 h later (pLH Ovsynch), or similar treatment plus a CIDR-B device from Days 0 to 7 (pLH Ovsynch + CIDR-B), or 1 mg estradiol benzoate (EB) and 100 mg progesterone on Day 0, a CIDR-B device from Days 0 to 7 (EB/ P4 + CIDR-B), PGF on Day 7 (at the time of CIDR-B removal) and 1 mg i.m. EB on Day 8 with AI on Day 9 (52 h after PGF). Pregnancy rate in the EB/P4 + CIDR-B group (75.0%) was higher (P < 0.04) than in the pLH Ovsynch group (37.5%): the pLH Ovsynch + CIDR-B group was intermediate (64.7%). In Experiment 4, 266 non-lactating cows were allocated to a 7-day Co-Synch protocol (Co-Synch), a 7-day Co-Synch plus 0.6 mg per head per day melengestrol acetate (MGA) from Days 0 to 6 inclusive (Co-Synch + MGA) or MGA (Days 0-6) plus 2 mg EB and 50 mg progesterone on Day 0. 500 microg PGF on Day 7, 1 mg EB on Day 8 and fixed-time Al 28 h later (EB/ P4 + MGA). Pregnancy rates (P < 0.25) were 44.8% (39/87: Co-Synch), 47.8% (43/90; Co-Synch + MGA), and 60.7% (54/89: EB/P4 + MGA). In conclusion, a 6- or 7-day interval from GnRH to PGF in a Co-Synch regimen resulted in similar pregnancy rates in cows. The addition of a progestin to a Co-Synch or Ovsynch regimen significantly improved pregnancy rates in heifers but not in cows. Progestin-based regimens that included EB consistently resulted in high pregnancy rates to fixed-time Al.     

Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows

The purpose of this study was to examine the effects of level of rumen inert fatty acids on developmental competence of oocytes in lactating dairy cows. Estrous cycles were synchronized in 22 cows on a silage-based diet supplemented with either low (200 g/day) or high (800 g/day) fat. A total of 1051 oocytes were collected by ultrasound-guided ovum pickup (OPU) in seven sessions/cow at 3-4 day intervals. Oocytes were matured, fertilized, and cultured to the blastocyst stage in vitro. Embryo quality was assessed by differential staining of Day 8 blastocysts. The high-fat diet reduced numbers of small and medium follicles. There was no effect on the quality of oocytes (grades 1-4) or cleavage rate. However, high fat significantly improved blastocyst production from matured (P < 0.005) and cleaved (P < 0.05) oocytes. Blastocysts from the high-fat group had significantly more total, inner cell mass and trophectoderm cells than the low-fat group (P < 0.05). Regression analysis showed negative effects of milk yield (P < 0.001), dry matter intake (P < 0.001), metabolizable energy intake (P < 0.005), and starch intake (P < 0.001) on blastocyst production in the low-fat group but not in the high-fat group. Within the low-fat group, blastocyst production was negatively related to growth hormone (P < 0.05) and positively related to leptin (P < 0.05). The low-fat group had higher nonesterified fatty acids than the high-fat group (P < 0.05). In conclusion, higher milk yields were associated with reduced developmental potential of oocytes in cows given a low-fat diet. Provision of a high-fat diet buffered oocytes against these effects, resulting in significantly improved developmental potential.     

Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen

The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose). Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 8-12 of the estrus cycle. Inseminations began 12h after the onset of estrus and were performed two to four times at 9-15 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen was placed into the uterine body. In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. The proportion of transferable female embryos produced approximately doubled when insemination was with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%; cows 81.1% versus 39.8%). In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermatozoa produced significantly smaller proportions of transferable embryos (p<0.005) and significantly larger proportions of unfertilized oocytes (p<0.001) than those inseminated with unsorted semen. Proportions of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated oocytes/embryos. It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for the insemination of superovulated embryo donors can improve the proportion of transferable female embryos produced but this potential may not be achieved in commercial practice, particularly in cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.     

Collection of oocytes and production of blastocysts in vitro from individual, slaughtered cows.

On four occasions ovaries from a total of 35 cows were collected separately at the abattoir where they had been killed. The age of 20 of these cows was recorded. Oocytes from these ovaries were collected separately and were submitted to in vitro maturation, in vitro fertilization and in vitro culture procedures. Ovaries of 34 randomly chosen cows were pooled and treated as the control. Ova from individual cows were cultured in 10 microliters droplets and those from pooled ovaries were cultured in groups of 50 in 50 microliters droplets of oviductal cell-conditioned medium. The 35 cows treated individually supplied 493 oocytes (mean 14.1 oocytes per cow) with high individual variation (SD = 10.0; range = 0-38) and 47 expanded blastocysts (9.5% of oocytes; mean 1.3 blastocysts per cow; range = 0-6). Among these cows, 16 produced one or more blastocysts. Considerable variation in average development rates was detected over the four replicate experiments (11.3, 4.0, 9.0 and 13.5%). The 34 cows treated as the control supplied 397 oocytes (mean 11.7 oocytes per cow) and 44 expanded blastocysts (11.1% of oocytes; mean 1.3 blastocysts per cow) with high variations between replicates (11.1, 4.0 and 18.1%). No difference was observed between individual and pooled ovaries regarding either the number of oocytes, the rate of blastocyst formation, or the number of blastocysts per cow. No effect of age was detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP).Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage.Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B.Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.     

Feeding frequency has diet-dependent effects on plasma hormone concentrations but does not affect oocyte quality in dairy heifers fed fibre- or starch-based diets

The post-fertilisation developmental capacity of bovine oocytes recovered by ultrasound guided transvaginal follicular aspiration (ovum pick-up, OPU) is influenced by diet-induced changes in hormone and metabolite concentrations. The objectives of this experiment were first to determine whether post-prandial changes in hormone concentrations, induced by changing the frequency of feeding, influenced oocyte quality and second whether changes in plasma glucagon concentration were associated with oocyte quality. Using a 2 × 2 factorial design, Holstein heifers (six per treatment) were fed either fibre- or starch-based diets containing either 189 or 478 g starch/kg dry matter. The diets were offered in either two or four equal meals per day and supplied twice the maintenance energy requirement. Blood samples were obtained both at weekly intervals (three samples per heifer, collected before feeding) during the experiment and throughout an entire 24-h period (15 or 17 samples per heifer for twice or four times daily-fed heifers, respectively). Each heifer underwent six sessions of OPU (twice weekly) beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviductal fluid following in vitro fertilisation. Mean weekly plasma insulin concentrations did not differ between diets, but plasma glucagon concentrations were greatest when heifers were fed the starch-based diet twice daily compared with the other diets. When heifers were offered four meals per day, there were no meal-related changes in hormone concentrations. However, when heifers were offered two meals per day, plasma insulin concentration increased after feeding the starch-based, but not the fibre-based diet. Plasma glucagon concentration increased after meals when heifers were fed twice daily and the increase was substantially greater when the starch-based diet was fed. Treatments did not influence (overall mean with mean ± s.e.) ovarian follicle size distribution or oocyte recovery by OPU (6.2 ± 0.4 per heifer), the proportion of oocytes that cleaved following insemination (0.57 ± 0.030) or blastocyst yield (0.27 ± 0.027 of oocytes cleaved). In conclusion, by feeding diets differing in carbohydrate source at different frequencies of feeding, meal-related changes in plasma hormone profiles were altered significantly, but oocyte quality was not affected. Therefore effects of diet on oocyte quality appear not to be mediated by meal-related fluctuations in hormone concentrations.