W.E. Beal

Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per aspïration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.

Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle

The objectives of this experiment were to determine the effects of corpus luteum characteristics, progesterone concentration, donor-recipient synchrony, embryo quality, type, and developmental stage on pregnancy rates after embryo transfer. We synchronized 763 potential recipients for estrus using one of two synchronization protocols: two doses of PGF2alpha (25 mg i.m.) given 11 d apart (Location 1); and, a single norgestomet implant for 7 d with one dose of PGF2alpha (25 mg i.m.) 24 h before implant removal (Location 2). At embryo transfer, ovaries were examined by rectal palpation and ultrasonography. Of the 526 recipients presented for embryo transfer, 122 received a fresh embryo and 326 received a frozen embryo. Pregnancy rates were greater (P < 0.05) with fresh embryos (83%) than frozen-thawed embryos (69%). Pregnancy rates were not affected by embryo grade, embryo stage, donor-recipient synchrony, or the palpated integrity of the CL. Corpus luteum diameter and luteal tissue volume increased as days post-estrus for the recipients increased. However, pregnancy rates did not differ among recipients receiving embryos 6.5 to 8.5 days after estrus (P > 0.1). There was a significant, positive simple correlation between CL diameter or luteal tissue volume and plasma progesterone concentration (r = 0.15, P < 0.01 and r = 0.18, P < 0.01, respectively). There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality.

The evaluation of corpus luteum blood flow using color-flow Doppler ultrasound for early pregnancy diagnosis in bovine embryo recipients

The objective of this experiment was to evaluate corpus luteum blood flow (CLBF) as an early indicator of pregnancy status in bovine embryo recipients. Fifty crossbred beef cows were submitted to embryo transfer on Day 7 after estrus. On Days 7, 11, 13, 15, 17, 19, 21, 26, 33, and 40, a blood sample was taken, the CL examined using a color-flow Doppler ultrasound scanner, and video was recorded of each scanning session. Ultrasound data were grouped by the first day progesterone concentrations were <1 ng/mL (indicating early embryo loss, EEL) through Day 21 (EEL-17, n=3; EEL-19, n=9; EEL-21, n=3), absence of an embryo on Days 26, 33, or 40 (late embryo loss; LEL; n=12), or remained pregnant (P; n=23). The first decrease in CLBF of EEL-17, EEL-19, and EEL-21 cows compared to P cows occurred on Days 17, 19, and 21, respectively (P<0.05). There was no difference in CLBF between LEL and P cows on Days 17, 19, and 21. Six evaluators diagnosed pregnancy from randomized video clips on Days 17, 19, and 21. Evaluators made more (P<0.004) correct diagnoses on Day 19 than Day 17. Sensitivity (82.9+/-10.1%) was not affected by day. From Days 17 to 19, diagnostic specificity increased (P=0.046) from 43.2+/-3.0 to 54.3+/-3.0% but remained unchanged thereafter. Due to low specificity and sensitivity, evaluation of CLBF alone was insufficient for early pregnancy diagnosis.

Association of prolactin and insulin-receptors with mammogenesis and lobulo-alveolar formation in pregnant ewes

1. Pregnant, multiparous ewes were sacrificed at d 50 (n = 3), 80 (n = 4), 115 (n = 3), or 140 (n = 4) for biochemical, histological and autoradiographical quantitation of mammary growth. 2. Significant increases in concentration of mammary parenchyma DNA (0.25-24 mg/g tissue), total under DNA (57-1304 mg), and total under RNA (36-1504 mg) were observed by d 115. 3. Mammary tissue at d 115 contained the maximal percent of tissue volume occupied by epithelium (41.2%), number of cells per alveolar cross section (36.6) and percent of [methyl-3H]thymidine labeled epithelial cell nuclei (3.5%). 4. Concentration prolactin binding sites were significantly increased at d 115 of gestation, but serum prolactin and growth hormone concentrations remained low until d 140. 5. Mammary insulin binding sites (per unit membrane protein) progressively decreased during gestation. 6. Results suggest that serum growth hormone concentration and quantity of insulin receptors do not limit mammogenesis but greater concentrations of prolactin binding sites coincident with lobulo-alveolar formation, suggest a primary association with mammogenesis in the ewe.

Evaluating embryo freezing method and the site of embryo deposition on pregnancy rate in bovine embryo transfer

Direct transfer of embryos frozen in ethylene glycol could lead to the increased practice of embryo transfer (ET) by an owner/herdsman. Two experiments were conducted to assess the potential of direct transfer with a specific effort to determine the importance of the “depth” of embryo placement in the uterus on pregnancy rate. In Exp. I 965 Grade 1 or 2 embryos were thawed for direct transfer after using ethylene glycol as a cryoprotectant or were thawed and rehydrated after being frozen in 10% glycerol. The embryos were frozen by 13 different ET companies, but were transferred to synchronous recipients by a single practitioner. The pregnancy rate was not different (Chi-square; P>.05) following transfer of embryos frozen in ethylene glycol (59.6%; 465/780) or glycerol (56.8%; 105/185). In Exp. II 68 Grade 1 or 2 embryos frozen in ethylene glycol were transferred directly to synchronous recipients by one practitioner. Recipients were randomly assigned to receive an embryo placed in the uterine horn ipsilateral to the corpus luteum at a point adjacent to the external bifurcation of the uterine horns (shallow) or at a point more than two-thirds of the distance from the external bifurcation toward the oviductal end of the uterine horn (deep). Placement deep in the uterine horn required manual manipulation to temporarily “straighten” the uterine horn from its coiled position typical of diestrus. Pregnancy rate was greater (P<.01) when embryos were placed deep in the uterine horn (see Table), regardless of whether embryos were placed in the left (+42.5%) or right horn (+33.3%) and regardless of embryo quality.

Application of knowledge about corpus luteum function in control of estrus and ovulation in cattle

Understanding corpus luteum (CL) function has led to development of methods of estrus synchronization in cattle that either extend the estrous cycle by administration of exogenous progestins or shorten the cycle by induction of luteolysis. Both methods have limitations, which have been reduced or overcome through sequential or combined treatment with progestin and luteolytic drugs. Future improvements in estrus synchronization methodology are most likely to come from achieving more synchrony between the development of a highly fertile ovulatory follicle and control of luteal function.

Reproductive responses of beef heifers after concurrent administration of vaccines, anthelmintic and progestogen

A study was conducted to determine whether concurrent administration of vaccines for reproductive diseases and an anthelmintic, given immediately prior to estrus synchronization with a progestogen, would affect subsequent estrous response and fertility in beef heifers. Two hundred ninety-five yearling crossbred beef heifers on 3 ranches in Oregon and Kansas were assigned to 1 of 2 treatments. Control heifers were vaccinated and dewormed 30 d prior to the anticipated breeding. Treated heifers received the same vaccination and deworming products 9 d prior to breeding. All heifers in both groups were treated with norgestomet (Syncro-Mate-B) for 9 d to synchronize estrus. The administered vaccine consisted of modified live virus for infectious bovine rhinotracheitis, parainfluenza and bovine viral diarrhea, with 5-way bacterin for leptospirosis and bacterin for vibriosis. Heifers were also administered ivermectin or fenbendazole for deworming. Heifers exhibiting synchronized estrus were artificially inseminated and subsequently exposed to fertile bulls. Pregnancy status was determined at a later date by palpation per rectum. Vaccination and deworming at the time of treatment with progestogen compared with immunizing and deworming 30 d prior to breeding did not impair the expression of behavioral estrus. The percentage of treated heifers conceiving to AI or natural service did not differ from that of the respective control heifers. These data suggest that vaccination, deworming and synchronization of estrus with norgestomet can be performed simultaneously without any detrimental effects upon occurrence of behavioral estrus or fertility.

Isolation of a population of intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin gradient

This study was conducted to determine the efficacy of isolating intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin (BSA) gradient. Either 0.25, 0.5, 1, 2.5 or 5x10(9) spermatozoa extended to 26 ml with Kiev extender were layered on a discontinuous BSA gradient (4% BSA over 10% BSA) contained within a 500-ml separatory funnel. After 1 h of sperm migration at 23 degrees C, four 30-ml aliquots designated Fractions 1, 2, 3, 4 and Fraction T were collected from the bottom of the funnel. Fraction 4 was the bottom fraction and Fraction T was the remnant of the applied sample. For all concentrations of applied spermatozoa, the percentage of progressively motile spermatozoa was greater in Fraction 4 than in Fractions T or 1 (P<0.05). Fraction 4 contained a greater (P<0.05) proportion of spermatozoa with normal apical ridge acrosomes than Fraction T. Regardless of the concentration of spermatozoa applied, Fraction 4 contained more than 90% of progressively motile spermatozoa and spermatozoa possessing normal apical ridge acrosomes. The percentage of applied cells recovered in Fraction 4 decreased as the concentration of spermatozoa applied to the gradient increased.

Prostaglandin F2α and naloxone therapy in the anestrous postpartum beef cow

Three experiments were conducted, using multiparous crossbred beef cows, to test the ability of exogenous prostaglandin F(2alpha) (PGF) and/or naloxone to reduce the duration of the postpartum interval to estrus and to improve subsequent reproductive performance. In each experiment, postpartum cows were assigned to treatments by calving date. In Experiment 1, cows (n=44) were assigned to 1 of 4 treatment groups: 1) control, 2) PGF on Day 25 post partum, 3) 400 mg naloxone (3 doses) at 12-h intervals on Day 30 post partum, and 4) PGF on Day 25 followed by 3 400-mg doses naloxone at 12-h intervals on Day 30 post partum. In Experiment 2, cows (n=126) were assigned either to 1) control or 2) PGF on Day 30 post partum In Experiment 3, cows (n=67) were again assigned to 1 of 4 treatments 1) control, 2) PGF on Day 30 post partum, 3) PGF on Day 40 post partum, and 4) PGF on Day 30 and 40 post partum. Serum progesterone was used to determine the postpartum interval to estrus in Experiments 1 and 3. In all 3 experiments, serum progesterone was used to determine the proportion of cows that had reestablished estrous cycles at the start of breeding. Pregnancy rate and calving interval were analyzed for all trials. Naloxone had no effect (P > 0.20) on any reproductive variable measured. The postpartum interval to estrus was similar (P > 0.30) for PGF-treated and control cows in Experiments 1 and 3. The proportion of cows cycling at the start of breeding and the calving interval were not affected (P > 0.20) by PGF treatment in any of the experiments. Only the administration of PGF on Day 40 post partum in Experiment 3 improved (P=0.04) the subsequent pregnancy rate. Analysis of data pooled across experiments showed that the pregnancy rate was higher (P=0.03) for cows treated with PGF than for control cows (91.4 and 72.9%, respectively). It was concluded that administration of PGF during the early postpartum period improves subsequent reproductive function in beef cows.