Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P < 0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells. (C) 2012 Elsevier B.V. All rights reserved.

Ovarian follicle diameter at timed insemination and estrous response influence likelihood of ovulation and pregnancy after estrous synchronization with progesterone or progestin-based protocols in suckled Bos indicus cows

The objectives of the present study were to evaluate factors associated with estrous synchronization responses and pregnancy per insemination (P/AI) in Bos indicus beef cows submitted to progesterone-based fixed-time artificial insemination (FTAI) protocols. A total of 2388 cows (1869 Nellore and 519 crossbred Nellore x Angus) from 10 commercial farms were evaluated to determine the relationships among breed, body condition score (BCS) on the first day of the FTAI protocol, the occurrence of estrus between progesterone device removal and FTAI and diameter of largest ovarian follicle (LF) at FTAI on estrous synchronization responses and P/AI. Cows (n=412 primiparous; 1976 multiparous) received an intravaginal device containing progesterone or an ear implant containing norgestomet (a progestin), and an injection of estradiol at the beginning of the estrous synchronization protocol. Body condition was scored using a 1-5 scale on the first day of the FTAI protocol and at 30-60 days postpartum. Females received 300IU of equine chorionic gonadotropin (eCG) and PGF(2 alpha) on the day the progesterone device/implant was removed and were inseminated 48-60h later. At insemination, cows (n=2388) were submitted to an ultrasonographic exam to determine the diameter of the LF. Follicles were classified into four categories based on mean and standard deviation (SD) of the LF (LF1 = two SD below the mean; LF2 = mean minus one SD; LF3 = mean plus one SD; LF4 = two SD above the mean). Ovulation rate was determined in a subset of cows (n=813) by three consecutive ultrasonographic exams: (1) at time of progesterone device/implant removal, (2) at time of FTAI and (3) 48 h after FTAI. Ovulation was defined as the disappearance of a large follicle (>= 8.0 mm) that was previously recorded. Estrus was determined in a subset of the cows (n = 445) by the activation of a detection of estrous patch placed on the tail head on the day of progesterone device/implant removal. Pregnancy was diagnosed 30 days after FTAI. Pregnancy was influenced (P = 0.001) by follicle diameter [LF1 = 27.5% (81/295), LF2 = 46.6% (328/705), LF3 = 57.9% (647/1118), LF4 = 63.3% (171/270)] and the occurrence of estrus [estrus = 67.7% (174/257) and no estrus = 36.2% (68/188)]. Follicle diameter at FTAI influenced ovulation rate [LF1 = 42.5% (34/80), LF2 = 73.9% (161/218), LF3 = 95.8% (407/425), LF4 = 97.8% (88/90)], the occurrence of estrus [LF1 = 54.8% (51/93), LF2 = 33.6% (43/128), LF3 = 68.9% (126/183), LF4 = 90.2% (37/41)] and P/AI among cows that had ovulations [LF1 =32.4% (11/34), LF2 = 50.3% (81/161), LF3 = 60.0% (244/407), LF4 = 68.2% (60/88)]. Improving estrous responses between progesterone device withdrawal and FTAI and increasing the diameter of the LF at FTAI may be important aspects to achieve improved estrous synchronization responses and P/AI following progesterone/progestin and estradiol based FTAI protocols in suckled Bos indicus cows. (C) 2010 Elsevier B.V. All rights reserved.

Effect of progesterone concentrations, follicle diameter, timing of artificial insemination, and ovulatory stimulus on pregnancy rate to synchronized artificial insemination in postpubertal Nellore heifers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows

Follicle diameter deviation is defined as the beginning of the differential change in growth rates between the largest and next largest follicles subsequent to wave emergence and is considered a key component of follicle selection. Follicle selection has been extensively studied in European breeds of cattle (Bos taurus) but has not been critically studied in Zebu breeds (Bos indicus). The objectives of the present study were to determine and compare the morphological characteristics of deviation associated with the first post-ovulatory wave (Wave 1) of the estrous cycle in Nelore heifers (n = 8) and nonlactating cows (n = 11). Beginning on the day of ovulation (day 0), the three largest follicles (F1-F3, respectively) were individually tracked every 12 h for 6 d using transrectal ultrasonography. In individual animals, deviation was determined graphically using visual inspection of the diameter profiles of F1, F2 and sometimes F3 (observed deviation) and mathematically using segmented regression analysis of the diameter differences between F1 and F2 or sometimes F3 (calculated deviation). Mean day of emergence of Wave 1 when F1 reached >3 rum (approximately 1 d after ovulation) and growth rate of F1 during deviation (approximately 1.4 mm/d) were not significantly different between heifers and cows. The results of determining the beginning of deviation within heifers and cows using the observed and calculated methods were not significantly different. Averaged over both methods, diameter deviation occurred 2.8 d after ovulation when F1 reached 5.7 mm in heifers, and 2.4 d after ovulation when F1 reached 6.1 mm in cows. In conclusion, the emergence of Wave 1 and growth rates and diameters of the future dominant follicles at the beginning of deviation were similar in Nelore heifers and nonlactating cows, regardless of the methods used to determine deviation. Relative to Holstein cattle, emergence of Wave 1 appeared to occur about 1 d later and diameter of the future dominant follicle at the beginning of deviation was about 2 turn smaller in Nelore. (C) 2004 Elsevier B.V. All rights reserved.

Expression of LH receptor mRNA splice variants in bovine granulosa cells: Changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroiclogenic ability, and in granulosa cells from small follicles (< 6 mm diameter) undergoing differentiation in vitro. Serniquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles < 7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles > 7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.

The effect of follicle age on pregnancy rate in beef cows

The effect of the age of the ovulatory follicle on fertility in beef cows was investigated. Multiparous (n = 171) and primiparous (n = 129) postpartum beef cows in 2 groups (G1 and G2) received estradiol benzoate (EB; 1 mg/500 kg BW, intramuscular [i.m.]) 5.5 d (G1; n = 162) and 6.5 d (G2; n = 138) after the final GnRH of a synchronization program (5d CO-Synch + CIDR) to induce emergence of a new follicular wave (NFW), followed by prostaglandin F2 alpha (PGF2 alpha; 25 mg, i.m.) administration either 5.5 d (young follicle, YF; n = 155) or 9.5 d (mature follicle, MF; n = 145) after EB. Estrous detection coupled with AI 12 h later (estrus-AI) was performed for 60 h (MF) and 84 h (YF) after PGF(2 alpha); cows not detected in estrus within this period received timed AI (TAI) coupled with GnRH at 72 and 96 h, respectively. Within the first 72 h after PGF(2 alpha), more (P < 0.01) cows in the MF (76.3%) than YF treatment (47.7%) exhibited estrus, but through 96 h, the proportion detected in estrus (P < 0.05) and interval from PGF(2 alpha) to estrus (P < 0.01) were greater in the YF than MF treatment (88.6% vs. 76.3%, 78.9 +/- 0.8 vs. 57.5 +/- 1.6 h, respectively). Age of the ovulatory follicle at AI was greater (P < 0.01) in the MF (9.32 +/- 0.04 d) than YF (6.26 +/- 0.02 d) treatment, but follicle diameter at AI and pregnancy rates did not differ between MF (13.1 +/- 0.2 mm; 72.0%) and YF (12.9 +/- 0.1 mm; 67.1%) treatments. Regardless of treatment, the diameter of the ovulatory follicle at AI and pregnancy rate were greater (P < 0.01) with estrus-AI (13.1 +/- 0.1 mm; 75.0%) than TAI (12.6 +/- 0.2 mm; 55.4%). Cows in the MF treatment that initiated a second NFW after EB but before PGF(2 alpha) (MF2; n = 47) were induced to ovulate with GnRH and TAI at 72h, when ovulatory follicles were 4 d old and 10.2 +/- 0.2 mm in diameter. Pregnancy rate for TAI (51.1%) in MF2 did not differ from TAI pregnancy rate (55.4%) across the MF and YF treatments. In summary, the age of the ovulatory follicle affected interval to estrus and AI but did not influence pregnancy rate in suckled beef cows.

Strategies to improve fertility in Bos indicus postpubertal heifers and nonlactating cows submitted to fixed-time artificial insemination

Two experiments were designed to evaluate strategies to increase fertility of Bos indicus postpubertal heifers and nonlactating cows submitted to a fixed-time artificial insemination (TAI) protocol consisting of an intravaginal device containing 1.9 g of progesterone (CIDR) insertion + estradiol benzoate on Day 0, CIDR withdrawal + estradiol cypionate on Day 9, and TAI on Day 11. In Experiment 1, heifers (n = 1153) received a new or an 18-d previously used CIDR and, on Day 9, prostaglandin F(2 alpha) (PGF(2 alpha)) + 0, 200, or 300 IU equine chorionic gonadotropin (eCG). Heifers treated with a new CIDR had greater (least squares means +/- SEM) serum concentration of progesterone on Day 9 (3.06 +/- 0.09 ng/mL vs. 2.53 +/- 0.09 ng/mL; P < 0.05) and a smaller follicle at TAI (11.61 +/- 0.11 nim vs. 12.05 +/- 0.12 mm; P < 0.05). Heifers with smaller follicles at TAI had lesser serum progesterone, concentrations on Day 18 and reduced rates of ovulation, conception, and pregnancy (P < 0.05). Treatment with eCG improved (P < 0.05) follicle diameter at TAI (11.50 +/- 0.10 mm, 11.90 +/- 0.11 mm, and 12.00 +/- 0.10 mm, for 0, 100, and 200 IU, respectively), serum progesterone concentration on Day 18 (2.77 +/- 0.11 ng/mL, 3.81 +/- 0.11 ng/mL, and 4.87 +/- 0.11 ng/mL), and rates of ovulation (83.8%, 88.5%, and 94.3%) and pregnancy (41.3%, 47.0%, and 46.7%). In Experiment 2, nonlactating Nelore cows (n = 702) received PGF(2 alpha) treatment on Days 7 or 9 and, on Day 9, 0 or 300 IU cCG. Cows receiving PGF(2 alpha) on Day 7 had lesser serum progesterone concentrations on Day 9 (3.05 +/- 0.21 ng/mL vs. 4.58 +/- 0.21 ng/mL; P < 0.05), a larger follicle at TAI (11.54 +/- 0.21 mm vs. 10.84 +/- 0.21 mm; P < 0.05), and improved (P < 0.05) rates of ovulation (85.4% vs. 77.0%), conception (60.9% vs. 47.2%), and pregnancy (52.0% vs. 36.4%). Treatment with eCG improved (P < 0.05) serum progesterone concentration on Day 18 (3.24 +/- 0.14 ng/mL vs. 4.55 +/- 0.14 ng/mL) and the rates of ovulation (72.4% vs. 90.0%) and pregnancy (37.5% vs. 50.8%). In conclusion, giving PGF(2 alpha) earlier in the protocol in nonlactating cows and eCG treatment in postpubertal heifers and nonlactating cows improved fertility in response to a TAI (progesterone + estradiol) protocol. (C) 2009 Elsevier B.V. All rights reserved.

Reproductive performance of prepubertal Bos indicus heifers after progesterone-based treatments

The objective was to evaluate the effects of exogenous progesterone (P4) on reproductive performance of prepubertal Bos indicus heifers. Prepubertal Nelore heifers (n = 589; 24.0 +/- 1.13 mo; 298.0 +/- 1.89 kg; body condition score of 3.2 +/- 0.26; mean +/- SEM) were randomly assigned to receive, between experimental Days -12 and 0: no treatments (CIDR0; n = 113); a new intravaginal insert (CIDR) containing 1.9 g of P4 (CIDR1; n = 237); or a similar insert previously used three times, with each use occurring for 9 d (CIDR4; n = 239). An additional treatment group was pubertal heifers given 12.5 mg dinoprost tromethamine im on Day 0 (PGF; n = 346), and used as controls for evaluation of conception rates. on Day 0, transrectal palpation was done for uterine score evaluation (UtS; 1-3 scale), blood samples were taken for serum P4 concentrations, and follicle diameter (FD) was measured. The breeding season started on Day 1 and consisted of AI after detection of estrus between Days I and 45, and exposure to bulls between Days 46 and 90. There were effects of treatment (P < 0.05) on serum concentrations of P4 on Day 0 (0.37 +/- 0.16, 2.31 +/- 0.11, and 1.20 +/- 0.11 ng/mL for CIDR0, CIDR1, and CIDR4, respectively; mean SEM), FD on Day 0(9.45 +/- 0.24, 9.72 +/- 0.17, and 11.42 +/- 0.16 mm), UtS on Day 0 (1.49 +/- 0.06, 1.88 +/- 0.04, and 2.24 +/- 0.04), estrus detection rates at 7 d (19.5, 42.6, and 38.3%) and 45 d (52.2, 72.1, and 75.3%) of the breeding season, and on pregnancy rates at 7 d (5.3, 14.3, and 18.4%), 45 d (27.4, 39.2, and 47.7%) and 90 d (72.6, 83.5, and 83.7%) of the breeding season. Conception rate 7 d after the start of the breeding season was greater (P < 0.05) in heifers from the CIDR4 (46.8%) and PGF (43.8%) groups than in the CIDR0 (27.3%) and CIDR1 (33.7%) groups. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. Furthermore, previously used CIDR inserts were better than new inserts. (C) 2010 Elsevier B.V. All rights reserved.

Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F-2 alpha treatment affect fertility in postpuberal Nelore heifers

Two experiments were performed to test the hypothesis that elevated progesterone concentrations impair pregnancy rate to timed artificial insemination (TAI) in postpuberal Nelore heifers. In Experiment 1, postpuberal Nelore heifers (n = 398) received 2 mg estradiol benzoate (EB) and either a new progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) (first use) or a CIDR previously used for 9 d (second use) or for 18 d (third use) on Day 0, 12.5 mg prostaglandin F-2 alpha (PGF(2 alpha)) on Day 7, 0.5 mg estradiol cypionate (ECP) and CIDR withdrawal on Day 9, and TAI on Day 11. Largest ovarian follicle diameter was determined on Day 11. The third-use CIDR treatment increased largest ovarian follicle diameter and pregnancy rate. Conception to TAI was reduced in heifers with smaller follicles in the first- and second-use CIDR treatments, but not in the third-use CID treatment. In Experiment 2, postpuberal Nelore heifers received the synchronization treatment described in Experiment 1 or received 12.5 mg PGF2. on Day 9 rather than Day 7. In addition, 50% of heifers received 300 IU equine chorionic gonadotropin (eCG) on Day 9. Heifers were either TAI (Experiment 2a; n = 199) or Al after detection of estrus (Experiment 2b; n = 125 of 202). In Experiment 2a, treatment with cCG increased pregnancy rate to TAI in heifers that received PGF2. on Day 9 but not on Day 7 and in heifers that received a first-use CIDR but not in heifers that received a third-use CIDR. Treatments did not influence reproductive performance in Experiment 2b. In summary, pregnancy rate to TAI in postpuberal Nelore heifers was optimized when lower concentrations of cxogcnous progesterone were administered, and eCG treatment was beneficial in heifers expected to have greater progesterone concentrations. (C) 2009 Elsevier B.V. All rights reserved.

Reuse of norgestomet implants in an eCG-based superovulation protocol administered to Nelore (Bos taurus indicus) cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ingestão de concentrado e concentração plasmática de progesterona em vacas da raça Holandesa

Vacas da raça Holandesa não-lactantes (n=7), que receberam 4kg/dia de concentrado durante 28 dias e pasto ad libitum, foram sincronizadas com protocolo Ovsynch. No dia 0 (segunda aplicação de GnRH), as vacas foram distribuídas em dois grupos: as do grupo 2kg receberam 2kg/dia, e as do grupo 8kg receberam 8kg/dia de concentrado, oferecido duas vezes/dia. Os animais foram sincronizados novamente na fase 2. Aqueles que receberam 2kg de concentrado na primeira fase passaram a receber 8kg na segunda fase e vice-versa. As estruturas ovarianas foram avaliadas diariamente por ultra-sonografia. Amostras de sangue para dosagem de progesterona (P4), por radioimunoensaio, foram colhidas diariamente, antes do fornecimento do concentrado e quatro horas depois, até o dia 16 do ciclo estral. Não houve diferença entre os grupos quanto ao diâmetro do folículo ovulatório no dia 0 e ao diâmetro do maior folículo no dia 16. O diâmetro do corpo lúteo foi maior (P<0,05) no dia 16 nos animais do grupo 8kg. Observou-se interação (P<0,05) da quantidade de concentrado fornecida versus dia da colheita das amostras quanto à concentração plasmática de P4. A concentração plasmática de P4, imediatamente antes do fornecimento do concentrado e quatro horas depois, foi menor no grupo 8kg, após o dia 9 do ciclo estral.

Influência do diâmetro e da fase folicular sobre a competência in vitro de oócitos obtidos de novilhas da raça Nelore

Avaliou-se o efeito do diâmetro e da fase do desenvolvimento folicular sobre a competência de oócitos para a produção in vitro de embriões bovinos. A primeira onda folicular foi sincronizada com progestógeno por nove dias e 24 horas após a sua retirada aplicou-se LH. Os ovários foram recuperados 60h (G-60), 96h (G-96) e 108h (G-108) após a ovulação induzida pelo LH. Os folículos foram dissecados ou aspirados e medidos e os oócitos recuperados e submetidos à maturação, fecundação e cultivo in vitro. Os ovários do G-60 apresentaram mais oócitos viáveis (graus I, II e III) (96,6%). A taxa de clivagem teve efeito significativo sobre o diâmetro folicular, sendo maior nos oócitos oriundos de folículos classe 3 (>7mm). Na taxa de produção de blastocisto observou-se interação diâmetro versus fase de desenvolvimento folicular. A taxa de produção de blastocisto foi maior em oócitos obtidos de folículos com diâmetros <5mm (classe 1) no G-60 (64,5%), de 5-7mm (classe 2) no G-96 (33,3%) e >7mm (classe 3) no G-108 (50%). Conclui-se que o diâmetro e a fase de desenvolvimento folicular influenciam a competência oocitária para o desenvolvimento in vitro. Nos estádios iniciais da onda folicular a produção de blastocisto foi maior em oócitos de folículos pequenos; com o avanço da onda, a produção de blastocistos foi maior em oócitos obtidos de folículos maiores.

Efeito do número de inseminações artificiais por ciclo sobre a fertilidade de éguas inseminadas com sêmen eqüino diluído, resfriado a 20ºC e transportado

Cento e vinte ciclos estrais de cento e cinco fêmeas eqüinas da raça Mangalarga Marchador foram analisados com o objetivo de estudar o efeito do número de inseminações artificiais por ciclo sobre a fertilidade de éguas inseminadas com sêmen resfriado. Os ciclos foram acompanhados por palpação retal e rufiação, e as inseminações realizadas a cada 48 horas, a partir de um folículo de 30-35 mm de diâmetro até a ovulação, com sêmen diluído, resfriado a 20ºC e transportado. As éguas foram aleatoriamente distribuídas nos seguintes grupos: T1 - uma IA/ciclo, T2 - duas IA/ciclo e T3 - três ou mais IA/ciclo. As taxas de concepção ao primeiro ciclo foram, respectivamente, para T1, T2 e T3, de 47,83% (11/23), 70,00% (21/30) e 45,16% (14/31) e, após cinco ciclos, de 51,43% (18/35), 48,89% (22/45), e 47,50% (19/40) (P>0,05). O número de IA/ciclo não teve efeito sobre a fertilidade das éguas.

Efeito do número de inseminações artificiais sobre a fertilidade de éguas inseminadas com sêmen diluído, resfriado a 14ºC e transportado

Estudou-se o efeito do número de inseminações sobre a fertilidade de éguas inseminadas, três vezes/semana (segundas, quartas e sextas-feiras), com sêmen diluído, resfriado e transportado, de apenas um garanhão. As éguas foram inseminadas a partir da detecção, pela palpação retal, de um folículo de 3,0 a 3,5 cm de diâmetro, em um dos ovários, até a ovulação. Utilizaram-se para o transporte o sêmen diluído no diluidor leite desnatado-glicose e o conteiner Celle modificado, sendo a dose inseminante de 400 x 10(6) espermatozóides móveis, no momento da diluição final, pré-resfriamento. de acordo com o número de inseminações artificiais (IA) utilizadas/ciclo, os resultados de 148 ciclos, de 100 éguas, foram agrupados em: 1 IA, 2IA, 3 IA e 4 ou mais IA. A eficiência de prenhez foi de 4,29; 5,04; 5,67; e 3,43, para 1, 2, 3 e 4 ou mais IA, respectivamente. As características foliculares diferiram em relação à freqüência de inseminações com os maiores valores observados no grupo de éguas inseminadas quatro ou mais vezes. Concluiu-se que menor velocidade de crescimento folicular e maior diâmetro do folículo ovulatório estiveram associados a maior número de inseminações artificiais/ciclo. Na presença de uma concentração espermática adequada, o número de inseminações/ciclo não exerceu influência sobre a fertilidade.

LH and FSH concentration and follicular development in Nellore heifers submitted to fixed-time artificial insemination protocols with different progesterone concentrations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)