Nitric oxide concentrations, estradiol-17ß progesterone ratio in follicular fluid, and COC quality with respect to perifollicular blood flow in cows.

The objectives were to investigate relationships among concentrations of nitric oxide (NO), estradiol 17 beta (E2), and progesterone (P4) in follicular fluids (FF), and quality of cumulus oocyte complexes (COCs) with respect to perifollicular blood flow (FBF). In Experiment I, follicles (138) were classified according to the presence or absence of FBF (assessed with transvaginal Doppler ultrasonography) and diameter of follicles (small, 2-4 mm; medium, 5-8 mm; and large, ≥9 mm). Concentrations of NO in FF did not differ significantly among these size categories. However, NO concentrations in FF with FBF (54.4 ± 7.4 µmol/l) were higher (P<0.05) than in those without FBF (36.6 ± 4.1 µmol/l). There was a positive correlation (r=0.30, P<0.05) between NO concentrations and the E2:P4 in FF. Rate of E2 active (E2:P4 ≥ 1) follicles were numerically 1.2 (0.8-1.8) times higher in follicles with FBF (38.1%) compared to those without FBF (25.0%). Moreover, rates of E2 active follicles were 6.1 (0.7-55.2) and 1.3 (0.1-17.3) times higher (P<0.06) in large (43.3%) and medium (14.3%) compared to small follicles (11.1%), respectively. In Experiment II, quality of COCs from 2 to 8 mm follicles, obtained by transvaginal ovum pick up (OPU), was investigated with respect to FBF. Odds ratio to obtain higher quality COCs from follicles with FBF (47.1%) was 3.3 (1.1-9.6) fold higher (P<0.05) compared to those from follicles without FBF (14.6%). In conclusion, E2:P4, and NO concentrations in FF, as well as FBF, could be used to determine the functionality of ovarian follicles in cows. Moreover, determination of FBF could be useful to predict quality of COCs in cattle.

Changes in follicular blood flow and nitric oxide levels in follicular fluid during follicular deviation in cows.

Two experiments were conducted to test the hypothesis that there are dynamic changes in follicular blood flow during follicular deviation and that nitric oxide (NO) in follicular fluid (FF) plays a role in regulation of follicular blood flow. In Experiment I, follicular blood flow of the two largest follicles was monitored by using Power Doppler ultrasonography during follicular deviation in sixteen follicular waves during eight estrous cycles in eight cows. Blood flow did not differ (P>0.05) between the dominant follicle (DF) and the largest subordinate follicle (SF) until the beginning of the deviation of the follicular size, but was higher (P<0.05) in DF than in the largest SF one and two days after the beginning of diameter deviation in ovulatory (n=5) and atretic (n=11) waves; respectively. In Experiment II, FF was aspirated from DF and the largest SF on the day of diameter deviation (DF, n=6; SF, n=6) and two days later (DF, n=12; SF, n=9). Nitric oxide did not differ (P>0.05) between DF and the largest SF on the day of diameter deviation but, one or two days after observed diameter deviation NO concentrations were lower (P<0.01) in DF compared to the largest SF. On the day of diameter deviation and two days later E2 levels in FF were higher (P<0.01) in DF than in the largest SF. P4 concentrations in FF were higher (P<0.05) in DF than in the largest SF on the day of diameter deviation, but did not (P>0.05) differ two days later. E2/P4 ratio in FF was the same (P>0.05) in DF and the largest SF on the day of diameter deviation, but was higher (P<0.01) in DF than in the largest SF one or two days later. In conclusion, area of follicular blood flow of DF and the largest SF increased in parallel with follicular size during follicular deviation. Furthermore, there were relationships between changes in follicular blood flow, NO concentrations and E2/P4 ratio in FF following the beginning of diameter deviation in cattle.

Postpartum suppression of ovarian activity with a Deslorelin implant enhanced uterine involution in lactating dairy cows.

Holstein cows received, subcutaneously a non-degradable implant containing 5mg of the GnRH agonist Deslorelin (DESL) or no implant (CON) at 2+/-1 days postpartum (dpp). All cows were injected with PGF(2alpha) at 9 dpp. Previous pregnant (PPH) and non-pregnant uterine horns (PNPH) were determined by palpation per rectum. In Experiment 1, cows [DESL implant (n=10) and CON (n=9)] were examined by ultrasonography to record ovarian structures (23, 30 and 37 dpp) and uterine horn and cervical diameters (16, 23, 30 and 37 dpp). Uterine tone was scored before ultrasonography. Vaginoscopy was conducted just after ultrasonography examination to assess cervical discharge and color of the external cervical os. Blood samples were collected on a weekly basis for hormonal analyses. In Experiment 2, cows [DESL implant (n=77) and CON (n=70)] were palpated per rectum and vaginoscopy at 30 dpp for scoring of uterine tone, uterine horns, cervical diameter, and discharge. Blood samples were collected only at 9 dpp. In Experiment 1, DESL-implant-treated cows had more Class 1 follicles (P<0.01), less Class 2 (P<0.01) and Class 3 follicles (P<0.01) and no corpus luteum (CL) formation (P<0.01). In CON cows, six of nine animals had visible CL at 25+/-7 dpp. At 9 dpp plasma concentration of E(2), P(4) (P<0.01) and PGFM (P<0.05) were less in the DESL-implant treatment group. Diameter of PPH (P<0.01), PNPH (P<0.01) and cervix (P=0.08) were less in the DESL-implant treatment associated with greater uterine tone (P=0.07). The DESL-implant cows had a greater frequency of clear cervical discharge (P=0.09) and pink cervical os (P=0.06). In Experiment 2, plasma concentrations of PGFM were less at 9 dpp in DESL-implant treatment (P<0.01). Diameters of the PPH (P<0.01) and PNPH (P<0.01) were less and more uterine tone (P<0.01) in the DESL-implant treatment. Diameter of cervix and frequency of a cervical discharge score did not differ between treatments. Treatment with non-degradable Deslorelin (5mg) implant during postpartum: (1) suppressed ovarian follicular development, (2) enhanced physical involution of the uterus and cervix, (3) increased tone of the uterine wall, (4) decreased frequency of purulent cervical discharges, and (5) reduced inflammatory processes of the reproductive tract.

Reproductive responses following postpartum suppression of ovarian follicular development with a deslorelin implant during summer heat stress in lactating dairy cows.

The objective was to evaluate pregnancy rate to a timed artificial insemination (TAI) protocol in the autumn for cows treated with a non-degradable GnRH agonist implant (Deslorelin [DESL], 5mg) during the summer heat stress period compared with non-treated controls (CON). Cows were randomly assigned to receive or not a DESL implant within 1-4 days postpartum (dpp) twice weekly, from 25 June through 8 August 2001. All cows in DESL implant and CON treatments were injected with PGF(2alpha) 7 days after enrollment. Ultrasonography (US) monitored numbers of ovarian follicles and corpus luteum (CL) at approximately 10, 30, 35/36, 45/44, 56/55 and 66/63dpp, while DESL implants were in situ and concurrently CON, respectively. DESL implants were removed at two specific days, 28 August and 4 September. Cows had DESL implant in situ for a range of 28-67 days, depending on date of enrollment and implant removal. Within 61-100dpp, 31 days after implant removal, DESL implant and CON cows were initiated in a Presynch-Ovsynch and TAI protocol. Pregnancy was evaluated by US and palpation per rectum at 28 and 46 days after TAI, respectively. Plasma concentrations of progesterone were analyzed for sets of blood samples collected during the Presynch-Ovsynch and at TAI day followed 8 days later. Cows in the DESL-implant treatment had more (P<0.01) Class 1 (3-5mm) follicles, less (P<0.01) Class 2 (6-9mm), Class 3 (> or =10mm) follicles and CL compared with CON cows. Proportion of cows having initiated estrous cycles after calving was less (P<0.01) in the DESL-implant treatment (52.2%, 58/111) compared with CON (93.7%, 104/111) at the beginning of Ovsynch. Pregnancy rate to TAI was less (P<0.01) in the DESL implant (27.5%, 33/120) compared with CON (53.9%, 69/128). Pregnancy rate to TAI was less (P<0.01) in DESL-implanted cows that had initiated estrous cycles after calving (30.6%, 19/62) compared with CON (53.7%, 65/121) cows having initiated estrous cycles after calving. Furthermore, pregnancy rate was less (P<0.01) for cows having ovulations that had initiated estrous cycles after TAI in the DESL implant (39.1%, 18/46) compared with CON (62.1%, 54/87) treatments. Pregnancy losses from day 28 to day 46 of pregnancy did not differ between DESL implant (15.1%, 5/33) and CON (13.0%, 9/69) treatments. The DESL implant induced a delay in initiation of a new wave of follicular development during the postpartum-heat stressed period. The lesser pregnancy rate in the DESL-implant treatment group may be due to a pool of heat stress damaged follicles that were depleted in the control group with re-occurring follicle waves.

Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian, conceptus, and insulin-like growth factor system responses.

Nonlactating dairy cows were used to examine effects of bovine somatotropin (bST) on components of the insulin-like growth factor (IGF) system. Estrus was synchronized in cows with a Presynch + Ovsynch protocol and timed AI (TAI; n = 55) or not TAI (cycling, C; n = 23) on d 0 (time of synchronized ovulation). On d 0 and 11, cows received bST (500 mg) or no bST, and were sacrificed on d 17. Pregnancy rates were less in bST cows (27.2%, 9 of 33) than in controls (63.6%; 14 of 22). In contrast, conceptuses were larger in bST-treated cows (39.2 +/- 4.8 cm) than in controls (20 +/- 4.3 cm). Total interferon-tau in uterine luminal flushings (ULF) was greater in bST-treated cows (7.15 > 2.36 microg). Number of class 2 follicles (6 to 9 mm) was less in bST-C cows on d 7 and 16. On d 17, corpus luteum (CL) weight tended to be greater in bST-treated cows. Concentrations of progesterone were greater after d 10 in C than in pregnant (P) cows. In the ULF, IGF-binding protein-3 was greater in bST-P cows than in pregnant cows. A tendency for an increase in IGF-I hormone concentrations in the ULF was detected on d 17 in bST-treated and cyclic cows. Endometrial mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 increased in bST-C, but not in bST-P cows. Treatment with bST increased plasma concentrations of insulin, IGF-I, and growth hormone (GH). In conclusion, bST may have hyperstimulated plasma IGF-I and insulin to cause asynchrony between conceptus and uterus that was detrimental to pregnancy.

Induction of ovulation in nonlactating dairy cows and heifers using different doses of a deslorelin implant.

The objective of this study was to evaluate ovarian function after inducing ovulation with a deslorelin implant in nonlactating dairy cows and heifers. Cattle received GnRH on Day -9, and PGF2alpha on Day -2. On Day 0, in Experiment 1, cows received either 100 microg GnRH (Control), a 750 microg (DESLORELIN 750) or 1000 microg (DESLORELIN 1000) deslorelin implant. On Day 0, in Experiment 2, cows received 100 microg of GnRH or a 450 microg (DESLORELIN 450) deslorelin implant. In Experiments 1 and 2, cows received PGF2alpha on Day 16. Ultrasonography and blood sampling for plasma progesterone (P4) were used to monitor ovarian activity. On Day 0, in Experiment 3, heifers received either 100 microg of GnRH or 750 microg (DESLORELIN 750) deslorelin implant. On Day 16, all heifers received PGF2alpha. Blood samples were collected on Days 7, 13 and 16. In Experiments 1-3, deslorelin implants did not elevate plasma concentrations of P4 in a systematic manner during the late luteal phase. In Experiments 1 and 2, deslorelin implants decreased the size of the largest follicle and the number of Class II and III follicles. In Experiments 1 and 2, deslorelin-treated cows failed to ovulate by Day 28. In conclusion, deslorelin implants induced ovulation, stimulated development of a normal CL, and delayed follicular growth during the subsequent diestrus period. For future applications, the dose of the deslorelin implant will have to be adjusted, and if used for timed-inseminations, nonpregnant cows will have to be resynchronized to minimize delayed returns to estrus and ovulation.

Strategies to optimize reproductive efficiency by regulation of ovarian function.

Pregnancy rate to the Ovsynch protocol can be improved if cows are presynchronized (i.e., two PGF(2alpha) injections given 14 days apart and the second injection of PGF(2alpha) given 12 days prior to the first GnRH of the Ovsynch program) so that a greater proportion of cows during the Ovsynch protocol ovulate to the first GnRH injection and have a CL at PGF(2alpha) injection. Pregnancy rates were normal in anestrous cows (39.6%) if they ovulated to both injections of GnRH. Estradiol cypionate (ECP) can be used to replace GnRH to induce ovulation as a modification of the Presync-Ovsynch program (i.e., Presync-Heatsynch). Pregnancy rates after TI were 37.1+/-5.8% for Presync-Ovsynch compared to 35.1+5.0% for Presync-Heatsynch. Use of ECP to induce ovulation was an alternative to GnRH in which greater uterine tone, ease of insemination and occurrence of estrus, improved acceptance by inseminators. A GnRH agonist (Deslorelin; 750 microg) implant inserted at 48 h after injection of PGF(2alpha), as a component of the Ovsynch protocol, induced ovulation, development of a normal CL and delayed follicular growth until 24 d after implant insertion. Utilization of Deslorelin implants (450 microg and 750 microg) to induce ovulation compared to GnRH (100 microg) within the Ovsynch protocol resulted in 27 d pregnancy rates (GnRH 100 microg, 39%; Deslorelin implants 450 microg, 40% and 750 microg, 27.5%) with 12.7%, 5.0% and 9.5% embryonic losses by 41 d of pregnancy, respectively. Induction of an accessory CL with injection of hCG on day 5 after insemination improved conception rates by 7.1%. Bovine somatotrophin injected at first insemination following a Presync-Ovsynch program in cycling-lactating dairy cows increased 74 days pregnancy rates (57.1%>42.6%).

Use of estradiol cypionate in a presynchronized timed artificial insemination program for lactating dairy cattle.

Experiment 1 evaluated pregnancy rates when estradiol cypionate (ECP) was used to induce ovulation as part of a timed artificial insemination (TAI) protocol in comparison to Ovsynch for lactating dairy cows in Florida (n = 371) and Texas (n = 321). Cows were presynchronized with two injections of PGF2, (25 mg, im) given 14 d apart with TAI protocols beginning 14 d after the second injection of PGF20. The TAI protocols consisted of an injection of GnRH (100 microg, im) followed by PGF2alpha 7 d later. Then, cows either received an injection of GnRH (Treatment I, Ovsynch) at 48 h after PGF2alpha and inseminated 16 to 24 h later or received an injection of ECP (1 mg, i.m.) at 24 h after PGF2alpha, (Treatment II; Heatsynch) and inseminated 48 h later. In Florida, pregnancy rates after TAI were 37.1 +/- 5.8% for Ovsynch compared with 35.1 +/- 5.0% for Heatsynch. In Texas, pregnancy rates were 28.2 +/- 3.6% for Ovsynch and 29.0 +/- 3.5% for Heatsynch. Overall pregnancy rates did not differ between Ovsynch and Heatsynch treatments. In Experiment 2, estrus and ovulation times were determined in lactating dairy cows submitted to the Heatsynch protocol. Frequencies of detected estrus and ovulation after ECP were 75.7% (28/37) and 86.5% (32/37), respectively. Mean intervals to ovulation were 55.4 +/- 2.7 h (n = 32) after ECP and 27.5 +/- 1.1 h (n = 27) after onset of estrus. Estrus occurred at 29.0 +/- 1.8 h (n = 28) after ECP. It is recommended that any cow detected in estrus by 24 h after ECP injection be inseminated at 24 h and all remaining cows be inseminated at 48 h because 75% (n = 24/32) of the ovulations occurred between > or = 48 h to < or = 72 h after ECP. Synchronization of ovulation and subsequent fertility indicated that estradiol cypionate could be used to induce ovulation for successful timed insemination.

Effects of hormonal treatments on reproductive performance and embryo production.

Developments in the use of drugs to improve reproduction and embryo production have focused on estrus and ovulation synchronization protocols and embryonic survival. Protocols for synchronization of ovulation eliminate the need for detection of estrus and allow timed insemination of all cows enrolled. Various estrogenic, progestational, GnRH and PGF2 alpha-like drugs are used to synchronize follicle development, CL regression and induction of ovulation. Strategies are discussed to optimize such programs to maximize herd pregnancy rates. Use of bovine Somatotrophin (bST) in combination with the Ovsynch protocol resulted in increased pregnancy rates, indicating possible effects on oocyte and embryonic development. Treatment of embryo donor cows with bST reduced the proportion of unfertilized oocytes and increased the number of transferable embryos. Furthermore, bST increased pregnancy rate when given to the recipient. Sub-luteal plasma progesterone concentrations after insemination have been associated with lower pregnancy rates. Injection of hCG on day 5 post-insemination resulted in induction of an accessory CL, increased plasma progesterone concentrations and increased conception rates. Strategies involving the use of sustained GnRH agonists to enhance CL development and alter follicular development are considered for future programs to enhance pregnancy rates.

Effects of the persistent dominant follicle on the ability of follicle stimulating hormone to induce follicle development and ovulatory responses.

Three experiments were conducted to evaluate the effect of an induced first wave persistent dominant follicle on folliculogenesis and ovulatory responses induced by FSH. On d 6 of a synchronized estrous cycle (d 0 = estrus), cows were treated with a Syncromate-B implant and two injections of PGF2, (25 mg, 0700 h; 15 mg, 1900 h, i.m.). Cows in the control group retained a first-wave persistent dominant follicle, but in the aspirated group, the first-wave dominant follicle was removed via transvaginal aspiration on d 10 (d 0 = estrus). Beginning on d 12, cows received 32 mg of FSH-P i.m. in decreasing doses at 12-h intervals over a 4-d period. On d 15, the Syncromate-B implant was removed, and cows were ovariectomized (experiment 1, n = 8) or inseminated (experiment 2, n = 11) at 10 and 22 h after the onset of estrus. Cows in experiment 3 received a used controlled intravaginal drug releasing (CIDR) device and two injections of PGF2alpha (25 mg, 0700 h; 15 mg, 1900 h; i.m.) on d 6. On d 8, the first-wave dominant follicle was aspirated (n = 6) or left intact (n = 5), and FSH treatment was initiated (20 mg of Folltropin in decreasing doses at 12-h intervals over a 4-d period), and on d 10 the used CIDR device was removed from all cows. Ovarian follicle size and number were examined daily by ultrasonography from d 5 of the estrous cycle. The persistent dominant follicle increased in size from 10.7 mm on d 5 to 15.4 mm on d 10 (experiments 1 and 2), and from 9 mm on d 5 to 20.4 mm on d 11 (experiment 3). From d 11 to 14, the number of class 1 (2 to 5 mm) follicles was lower in the aspirated group than in the control group; the number of class 2 (6 to 9 mm) follicles was higher on d 12 and 13 for the aspirated group (experiments 1 and 2). The number of class 3 (> or =10 mm) follicles was higher in the aspirated group on d 14 to 16, but the same on d 17. Ovarian and embryo responses to superovulation did not differ between groups. In experiment 3, the numbers of class 1, 2, and 3 follicles, as well as ovarian and embryo responses following ovulation did not differ between groups. Initiation of exogenous FSH treatment appears to override any systemic inhibitory effect that a persistent dominant follicle may be exerting at the pituitary and possibly the ovary.