Oocyte and embryo production and quality after OPU-IVF in dairy heifers given diets varying in their n-6/n-3 fatty acid ratio

Dietary fat supplementation can improve oocyte quality in ruminants. The influence of the type of dietary fat on the number and quality of oocytes collected by ovum pick-up and on the production of embryos in vitro was investigated in Holstein heifers. Heifers were given hay plus one of two dietary supplements for 42 days. The supplements were linseed (L, rich in linolenic acid, C18:3n-3, n = 9) or soya bean (S, rich in linoleic acid, C18:2n-6, n = 9). Oocytes were collected by ovum pick-up (OPU) for 6 wks (2 sessions/wk) and morphologic quality assessed. Half the oocytes were frozen and the other half was used to produce embryos. Blood samples were analyzed for: insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids, β-hydroxy butyrate and urea and the proportions of fatty acids. Neither growth rate nor plasma hormone and metabolite concentrations were affected by dietary supplement. However, L significantly increased the proportion of plasma C18:3n-3 while S significantly increased the proportion of C18:2n-6(P < 0.001). Neither oocyte characteristics (number, their quality and number fertilized and cleaved per heifer per session) nor embryo characteristics (number and quality per heifer per session) and embryo development stages were affected by dietary treatment. Real-time RT-PCR was performed on immature and mature cumulus-oocyte complexes (COC). Prostaglandin E synthase-1 expression increased in L compared to S heifers. In conclusion, the type of fatty acid did not modify the numbers of oocytes and embryos produced by OPU-IVF and their quality in dairy heifers. Upregulation of prostaglandin E synthase-1 may ensure sufficient PGE₂ production for oocyte maturation even when its precursor is low.     

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.     

Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle

The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.     

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL ± 1.13 vs 22.12 mg/dL ± 0.86; mean ± SEM), neither the number of COCs recovered (8.8 ± 1.0 vs 9.2 ± 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes.     

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.     

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.     

Effects of circulating progesterone and insulin on early embryo development in beef heifers

The aims of this study were to determine the effect on early embryo development of feeding a diet formulated to enhance circulating insulin concentrations and secondly to investigate the association between early embryo development and maternal progesterone concentrations in beef heifers. The study was carried out in 32 Simmental x Holstein Friesian heifers 22-25 months of age weighing 506+/-7kg and in condition score 3.1+/-0.1. Animals were fed two diets that were isoenergetic and isonitrogenous, but that would encourage either propionate (diet A) or acetate (diet B) production in the rumen. The rationale was that propionate would induce a greater insulin release in response to feeding. Animals were fed a 50:50 mix of the two diets for 14 days at 0.8x maintenance, with straw provided ad libitum. Animals were then fed one of the experimental diets for 3 weeks prior to synchronisation of oestrus and insemination and for a further 16 days following mating. All heifers were blood sampled daily from oestrus synchronisation and eight animals on each diet underwent daily transrectal real-time ultrasonography to determine the day of ovulation. All heifers were slaughtered at Day 16 after mating. While feeding of diet A (propionic) caused a significant (P<0.05) increase in the plasma insulin to glucagons ratio differences in insulin were not significantly different. This is probably due to the fact that insulin concentrations were quite high as the heifers used in the present study were in good body condition making further increases in insulin difficult to achieve. Diet did not affect size of ovulatory follicle (DIET A: 15.1+/-0.7mm; diet B: 14.6+/-0.7mm), day of ovulation (diet A: 3.5+/-0.2 days; diet B: 3.4+/-0.2 days), mean plasma progesterone concentration (diet A: 4.7+/-0.4ng/ml; diet B: 5.2+/-0.3ng/ml), corpus luteum weight (diet A: 6.0+/-0.2g; diet B: 6.0+/-0.2g) or pregnancy rate (diet A: 81.3%; diet B: 81.3%). However, the proportion of well-elongated (>10cm) embryos on Day 16 was higher in animals fed diet A than in those fed diet B (84.6% versus 38.5%; P<0.05). While progesterone concentration did not differ between pregnant and non-pregnant heifers, progesterone did show an earlier post-ovulatory rise in heifers with well-elongated (>10cm) embryos with levels in these animals significantly higher on Days 4 and 5 than in heifers with small (<10cm) embryos at slaughter. This study demonstrated an enhancement in early embryo development in animals fed a diet generating an increased insulin:glucagon ratio that was not related to circulating maternal progesterone concentrations. However, across diets, enhanced embryo development was associated with elevated plasma progesterone on Days 4 and 5 following mating.     

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.     

Carbohydrate and amino acid metabolism and oxidative status in Holstein heifers precision-fed diets with different forage to concentrate ratios

Previous work led to the proposal that the precision feeding of a high-concentrate diet may represent a potential method with which to enhance feed efficiency (FE) when rearing dairy heifers. However, the physiological and metabolic mechanisms underlying this approach remain unclear. This study used metabolomics analysis to investigate the changes in plasma metabolites of heifers precision-fed diets containing a wide range of forage to concentrate ratios. Twenty-four half-sib Holstein heifers, with a similar body condition, were randomly assigned into four groups and precision fed with diets containing different proportions of concentrate (20%, 40%, 60% and 80% based on DM). After 28 days of feeding, blood samples were collected 6 h after morning feeding and gas chromatography time-of-flight/MS was used to analyze the plasma samples. Parameters of oxidative status were also determined in the plasma. The FE (after being corrected for gut fill) increased linearly (P < 0.01) with increasing level of dietary concentrate. Significant changes were identified for 38 different metabolites in the plasma of heifers fed different dietary forage to concentrate ratios. The main pathways showing alterations were clustered into those relating to carbohydrate and amino acid metabolism; all of which have been previously associated with FE changes in ruminants. Heifers fed with a high-concentrate diet had higher (P < 0.01) plasma total antioxidant capacity and superoxide dismutase but lower (P ≤ 0.02) hydroxyl radical and hydrogen peroxide than heifers fed with a low-concentrate diet, which might indicate a lower plasma oxidative status in the heifers fed a high-concentrate diet. Thus, heifers fed with a high-concentrate diet had higher FE and antioxidant capacity but a lower plasma oxidative status as well as changed carbohydrate and amino acid metabolism. Our findings provide a better understanding of how forage to concentrate ratios affect FE and metabolism in the precision-fed growing heifers.     

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.     

Effect of elevating serum lipids on luteinizing hormone response to gonadotrophin releasing hormone challenge in energy-deficient anestrous heifers

A study was conducted to evaluate the effect of feeding a bypass fat on luteinizing hormone (LH) response to gonadotrophin releasing hormone (GnRH) in noncyclic Holstein heifers. Twelve cyclic Holstein heifers were fed a complete diet at 40% net energy for maintenance (NE(m)) until cessation of ovarian activity. Based on weights and condition scores, heifers were assigned to either a control or treatment diet containing 0.45 kg bypass fat and fed at an energy level of 85% NE(m). Diet adjustments were made following weekly weighings. GnRH challenges were conducted at four periods: prior to initial energy deprivation, at termination of 40% NE(m) feeding, and twice more at 21-d intervals after 85% NE(m) feeding began. Blood was sampled via a jugular catheter every 15 min for 5 h, and GnRH was injected after the fourth sample. None of the heifers exhibited estrous activity after the initial energy deprivation. Heifers on the bypass fat diet continued to lose weight during the treatment period, while the control heifers gained a slight amount of weight. Baseline and peak concentrations of LH were not significantly affected by time or diet. Time to GnRH-induced LH peak was longer (53 vs 130 min, P < 0.01) after 40% NE(m) and remained greater at all times thereafter. Serum lipid levels increased 82.5% among heifers being fed the bypass fat. Energy restriction had no effect on the magnitude of LH response to GnRH but did delay response time.     

Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in simmental heifers

Transvaginal ovum pick-up (OPU) offers several advantages over standard embryo transfer procedures. For a systematic comparison of the ultrasound-guided (U-OPU) and the laparoscopic OPU (L-OPU) method, groups of Simmental heifers were subjected to 1 of the 2 OPU-methods for 8 wk (15 sessions) followed by a treatment-free interval of 11 wk and then another 8-wk period of OPU using the alternative method. Parameters that were evaluated included the number of follicles aspirated, number and morphology of recovered cumulus-oocyte complexes (COCs), and developmental capacity of oocytes after in vitro maturation (IVM) and fertilization (IVF). Blood samples were also taken from the donors twice a wk for progesterone measurement. To evaluate effects of long-term OPU on subsequent fertility of donors, all heifers (n = 14) were inseminated during the first natural estrus after the OPU experiment. The proportion of Class I oocytes was significantly (P < 0.001) higher after U-OPU than after L-OPU (38.7% vs 21.0%). Following IVM/IVF, this difference in oocyte quality was reflected by the cleavage rate (U-OPU: 58.1%; L-OPU: 52.1%; P < 0.05) and the rate of development to morulae and blastocysts (U-OPU: 27.1%; L-OPU: 13.9%; P < 0.001). Among other factors, the greater changes in vacuum pressure during L-OPU vs U-OPU might be responsible for the difference in oocyte quality. This problem may be overcome by a more flexible system for regulating the vacuum. Progesterone levels were higher during the L-OPU than the U-OPU periods. Seven donors (50%) were diagnosed pregnant by ultrasonography on Day 28 and by palpation per rectum on Day 42.     

Effects of diet type on establishment of pregnancy and embryo development in beef heifers

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18-24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5kg concentrates and 250g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250g feed grade urea and 50g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR(?), InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15mg luprostiol was given 12h before pessary withdrawal. They received 144mg pFSH (Folltropin(?)-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1±0.5, 4.9±0.3 and 3.2±0.1mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2±0.8, 5.8±1.5 and 6.8±1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7±0.6, 3.4±1.1 and 4.8±0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4±0.8 compared with 1.0±0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0±13.3, 86.9±7.2 and 76.5±7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3±11.8, 92.0±3.5 and 86.8±5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.     

Circulating gonadotrophins during a period of restricted energy intake in relation to body condition in heifers.

Beef heifers, 13 months old, were fed to achieve high (7.6 +/- 0.2 units) or low (3.9 +/- 0.1 units) body condition by feeding them one of two diets for 20 weeks. During week 17 of the growth phase, all heifers were ovariectomized. From week 20 to week 27 (restriction phase), all heifers were fed a daily diet containing 0.071 MJ metabolizable energy kg-1 body weight. At weekly intervals throughout the restriction phase, blood samples were collected at 10-min intervals for 11 h to determine the pattern of secretion of luteinizing hormone (LH), the amount of LH released in response to 750 ng (pituitary responsiveness) and 50 micrograms LH-releasing hormone (LHRH, releasable stores) and mean concentrations of follicle-stimulating hormone (FSH) in the circulation. Body weight declined during the restriction phase in a similar fashion in heifers with high and low body condition and changes in body weight were unrelated to mean concentrations of LH and FSH and frequency of LH pulses. Amplitude of LH pulses and responsiveness to 750 ng LHRH increased in a linear fashion with weight loss in heifers with low but not in those with high body condition. The amount of LH released in response to 50 micrograms LHRH decreased with increasing weight loss in heifers with high but not with low body condition, indicating that releasable pools of LH declined with increased weight loss in heifers with high body condition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH superstimulation

The objective in this study was to investigate the effect of repeated oocyte collection by transvaginal, ultrasound-guidance, oocyte pick-up (OPU) in nine, prepubertal (8-12 months), swamp buffaloes. Animals were treated with FSH for 3 days and received GnRH on the third day, 24 h before OPU. This session was repeated on five occasions at 2 weekly intervals. Over the five sessions of hormone treatment followed by OPU, 39/42 (92.9%) animals responded and had 6.6+/-3.6 follicles with a follicular diameter of 5.0+/-2.0 mm. The oocyte recovery rate was 5.4+/-3.7 and averaged 82.8% oocytes, except for session 4, when oocyte recovery was around 75.0%. Most oocytes were denuded (39.5%), whilst 28.8% had a substantial cumulus mass. There were no differences in the ovarian responses and the recovery rates between the collections. It was concluded that five repeat cycles of FSH and OPU did not influence the follicular response to superstimulation or the number of oocytes recovered from prepubertal, buffalo calves.     

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.     

Dietary energy source and feeding levels during the rearing period affect ovarian follicular development and oocyte maturation in gilts

Fifty-four Landrace × Yorkshire gilts (59.0 ± 4.2 kg and 147 ± 3 d old) were used to examine the effects of dietary energy source (starch or mixed fat) at high [112.5% of energy requirements recommended by NRC (1998)], normal (100%), and low (87.5%) energy feeding levels on ovarian follicular development and oocyte maturation. Forty-seven estrus gilts were slaughtered at Day 19 after the second estrus; oocytes were recovered from follicles >4 mm in diameter, and matured in vitro for 44 h. Gilts fed high-energy diets had more follicles >4 mm (mean, 25.8 vs. 19.1, P < 0.05) and more oocytes that reached metaphase II (80.3 vs. 64.0%, P < 0.05) than those fed the low-energy diet. Furthermore, gilts fed starch-rich diets had enhanced oocyte nuclear maturation relative to those fed fat-rich diets (75.4 vs. 68.0%, P < 0.05). Compared to the lower-energy feeding groups, high-energy feeding groups had higher (P < 0.05) blood concentrations of postprandial insulin (1562.4 vs. 990.0 ng/4 h), IGF-I (321.2 vs. 256.9 ng/mL), and LH pulses (2.7 vs. 1.4 pulses/6 h). Follicular fluid concentrations of IGF-I (198.5 vs. 143.1 ng/mL) and estradiol (152.6 vs. 124.8 ng/mL) were higher (P < 0.05) in the high-energy group than in the normal group. Compared with gilts fed the high-energy diet supplemented with fat, gilts fed the high-energy diet supplemented with starch had a tendency (P < 0.10) towards increased IGF-I concentration in both blood and follicular fluid, and improved oocyte nuclear maturation during culture in vitro. We inferred that starch-rich, high-energy diets during rearing may improve ovarian follicular development and oocyte maturation in replacement gilts.     

Insulin Sensitivity of Heifers on Different Diets

The hyperinsulinemic euglycemic clamp technique was used to investigate the effect on insulin sensitivity of 2 different diets used in practical cattle feeding in calves. Ten 4 to 5-month-old heifer calves were allocated to 2 feeding groups, LO or HI, to obtain growth rates of 400 g/day or 900 g/day. The heifers were fed and housed individually for 5 weeks. Growth rates close to calculated rates were obtained with the diets used. Weekly blood samples were collected from the jugular vein for analysis of glucose, insulin, cortisol, total serum protein, urea, cholesterol and nonesterified fatty acids. During week 5, insulin sensitivity was estimated using the hyperinsulinemic euglycemic clamp technique. Insulin sensitivity did not differ between the groups, but the plasma glucose levels were higher during weeks 3 and 4 for the HI group compared to the LO group. It may be concluded that the amount of concentrate in the diet was too low to induce changes in either the basal plasma insulin levels or the insulin sensitivity in the HI group.     

Metabolic hormone secretion and FSH-induced superovulatory responses of beef heifers fed dietary fat supplements containing predominantly saturated or polyunsaturated fatty acids

Ovulatory responses following FSH treatment were examined in beef heifers fed dietary fat supplements expected to produce differential effects on serum insulin concentrations and follicular recruitment patterns. Twenty-one heifers (n = 7/group) exhibiting regular estrous cycles were assigned randomly to either a control diet or to 1 of 2 fat-supplemented diets consisting of soybean oil (polyunsaturated fatty acids) or animal tallow (saturated fatty acids). The diets were formulated to be isoenergetic and isonitrogenous, and were fed until ovariectomy between experimental Days 35 and 45. Experimental Day 1 was defined for each heifer as the first day all of the treatment diet was consumed. After 20 d of diet consumption, estrous cycles were synchronized with prostaglandin F(2alpha) (PGF(2alpha)), and ovarian follicle populations were monitored via transrectal ultrasound for 4 d. Four days after estrus, the dominant follicle was aspirated and heifers were treated with FSH-P to induce superovulation. Ovulation rate was determined at ovariectomy 5 d after the superovulatory estrus (experimental Days 35 to 45). Both soybean oil and animal tallow diets increased (P < 0.05) the number of medium-sized follicles and increased (P < 0.02) serum concentrations of GH relative to the control diet. The soybean oil diet also increased (P < 0.001) serum concentrations of insulin on Days 14, 28, and 5 d after the superovulatory estrus. However, the number of ovulations following FSH treatment did not differ due to diet. Procedures employed in the current study were ineffective in recruiting the increased number of medium-sized follicles into the superovulatory pool.     

Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows

High plasma urea nitrogen (PUN) concentrations are associated with decreased fertility in lactating dairy cows. Our objective was to evaluate the quality of embryos flushed from superovulated lactating cows having moderate or high PUN concentrations. Subsequent embryo survival was determined after transfer to recipient heifers with either low or high PUN. Lactating Holstein dairy cows (n = 23; 50-120 days in milk) were randomly assigned to one of two diets designed to result in moderate or high PUN concentrations (15.5 +/- 0.7 and 24.4 +/- 1.0 mg/dl, respectively; P < 0.001) and were fed for 30 days before embryo flushing and recovery. Embryos (n = 94) were evaluated morphologically, frozen and subsequently transferred into synchronized virgin heifers that were fed one of two diets designed to result in either low or high PUN concentrations (7.7 +/- 0.9 and 25.2 +/- 1.5 mg/dl, respectively; P < 0.001; 2 x 2 factorial design). The number, quality and stage of development of recovered embryos were similar for cows with moderate or high PUN. Transfer of embryos from moderate PUN donor cows resulted in a higher pregnancy rate (35%; P < 0.02) than the transfer of embryos from high PUN donor cows (11%). Pregnancy rate was not affected by either recipient diet or the interaction of donor and recipient diets (P > 0.05). These results indicate that high PUN concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before recovery from the uterus 7 days after insemination.