Intrinsic and extrinsic factors that influence ovarian environment and efficiency of reproduction in cattle

The emergent concepts on ovary environment,reproductive physiology and the development ofpharmacology are constantly supporting the advance ofassisted reproduction. Within the last years, thebiotechnics related to the synchronization of folliculardevelopment and the manipulation of bovine estruscycle have progressed rapidly and consistently. Thecombined use of timed-artificial insemination (TAI),superovulation (SOV), ovum pick up (OPU), in vitroembryo production (IVEP) and timed-embryo transfer(TET) has a great potential to improve reproductiveoutcomes and disseminate selected genetics,diminishing the interval of generations and improvingherds genetic gain. However, several factors canpotentially affect the efficiency of these procedures. Theknowledge of the particularities of the genetic groups,follicular growth manipulation, follicular populationpredictors, and metabolic and environmental aspectsthat interfere with ovarian environment and,consequently, oocyte quantity and quality is crucial tooptimize the reproductive programs. This review aimsto elucidate some factors that affect the ovarianenvironment and must be well known in order toimprove the efficiency of reproduction in cattle.

Timed artificial insemination: current challenges and recent advances in reproductive efficiency in beef and dairy herds in Brazil

Beef and dairy productivity depends directly on the reproductive efficiency and genetic gain of the herd, which can be related to the appropriate use of biotechnologies, such as timed artificial insemination (TAI). When considering variations in synchronizations protocols, longer or shorter periods of progesterone (P4) device treatment could provide benefits to fertility. However, our studies evidenced that protocols with six (J-synch), seven, eight and nine days of P4 device treatment had similar pregnancy per AI (P/AI). In cyclic cows, the early prostaglandin (PGF) administration, moving from the day of P4 device removal to two days earlier, which results in four handlings of cows, or the administration of one extra dose of PGF at the onset of the protocol and a single PGF on the day of P4 device removal (three handlings) are both efficient to induce early luteolysis, reducing serum P4 concentrations and, therefore, stimulating LH pulsatility, which improves growth of the dominant follicle and results greater P/AI when compared with protocols with the administration of PGF only on the day of P4 device removal. Resynchronization is another valuable tool to reduce the interval between AI. Traditional Resynch is initiated at pregnancy diagnosis (28 to 32 days after TAI) and the interval between AI is around 40 days; Resynch 22 and Resynch 14 respectively initiates 22 and 14 days after the previous AI in all cows (unknown status of pregnancy) and reduces the interval between AI to 32 and 24 days. The novelty about Resynch 14 is the need to use of Doppler ultrasonography for pregnancy diagnosis [evaluation of corpus luteum (CL) vascularization].

Timed artificial insemination: current challenges and recent advances in reproductive efficiency in beef and dairy herds in Brazil

Beef and dairy productivity depends directly on the reproductive efficiency and genetic gain of the herd, which can be related to the appropriate use of biotechnologies, such as timed artificial insemination (TAI). When considering variations in synchronizations protocols, longer or shorter periods of progesterone (P4) device treatment could provide benefits to fertility. However, our studies evidenced that protocols with six (J-synch), seven, eight and nine days of P4 device treatment had similar pregnancy per AI (P/AI). In cyclic cows, the early prostaglandin (PGF) administration, moving from the day of P4 device removal to two days earlier, which results in four handlings of cows, or the administration of one extra dose of PGF at the onset of the protocol and a single PGF on the day of P4 device removal (three handlings) are both efficient to induce early luteolysis, reducing serum P4 concentrations and, therefore, stimulating LH pulsatility, which improves growth of the dominant follicle and results greater P/AI when compared with protocols with the administration of PGF only on the day of P4 device removal. Resynchronization is another valuable tool to reduce the interval between AI. Traditional Resynch is initiated at pregnancy diagnosis (28 to 32 days after TAI) and the interval between AI is around 40 days; Resynch 22 and Resynch 14 respectively initiates 22 and 14 days after the previous AI in all cows (unknown status of pregnancy) and reduces the interval between AI to 32 and 24 days. The novelty about Resynch 14 is the need to use of Doppler ultrasonography for pregnancy diagnosis [evaluation of corpus luteum (CL) vascularization].(AU)

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herd’s genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds Were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90’s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3).

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herds genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds Were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3).(AU)

Heat stress and embryo production in high-producing dairy cows

Background: Heat stress (HS) can compromise the female reproductive system, resulting in a massive reduction of reproductive performance. During the period of follicular growth, HS may compromise the oocyte, either because of a direct effect of elevated temperature on the gamete or because of changes in follicular function that would damage oocyte quality. Oocytes harvested from Holstein cows during summer have reduced ability to develop to the blastocyst stage than those harvested during winter. Nevertheless, although there are reports showing a clear effect of HS on oocyte competence to development after IVF, the exact mechanisms by which the oocyte is compromised remain unknown. Review: Resistance to HS is dependent on cow's genotype. Bos indicus breeds (i.e. Gir) are more resistant to elevated temperature and humidity than breeds (i.e. Holstein) that evolved in a temperate climate. This resistant is, among other factors, due to the superior ability of certain breeds to regulate their body temperature. Thus, breed selection is present as a possible tool to minimize the effects of temperature and humidity on milk production and reproduction. On the other hand, Holstein cows are the most common breed used, even in tropical countries (i.e. Brazil), because of their yield in milk production. As a result, there is a considerable effect upon milk production and reproductive performance through the year in tropical and subtropical countries. A possible way to minimize this effect is the employment of reproductive biotechnologies such as fixed-time artificial insemination (FTAI) and embryo transfer (ET). FTAI has been shown to eliminate problems of estrus detection caused by HS. Nevertheless, it is not enough to complete restore herd pregnancy rates because oocytes and early embryos have already been damaged by HS. In contrast, embryos three days after conception orolder are less sensitive to HS. Thus, ET has been used to improve cow's reproductive performance during the hot months of the year (i.e. spring and summer). This is carried out by producing embryos during the fresh months but using them for ET during the HS period. Regarding fertility problems, it is recognized that repeat-breeder (RB) cows cause huge economic problems to the farmer. These cows are characterized by poor fertilization rates and/or early embryonic loss. It is still not certainly known why these cows become RB, when it is the critical period of the year for them, and what should be done to avoid this problem. We have observed that the conception rates in RB Holstein cows were greater after ET (41.7%) than after AI (17.9%). This indicates that ET may be an effective alternative to achieve satisfactory conception rates throughout the year in RB cows, especially during periods of HS. Moreover, when three categories of Holstein cows (heifers, high-producing cows in peak lactation and RB) were subjected to ovum pick up during the summer and the winter, blastocyst rates and quality were significantly affected by both, category and period, being RB cows mostly affected during the summer. Moreover, lower amounts of mitochondrial DNA were found in RB oocytes, suggesting that the low reproductive performance observed in these cows is related to some injure in oocyte quality. Conclusion: HS negatively affects the physiology and fertility of dairy cows and heifers. Concerning about the reproductive field, the effects of HS were detected in vitro and in vivo on oocytes and embryos and also on conception rates. Moreover, RB Holstein cows seem to be even more sensitive to HS and this can probably be related to their oocyte potential into develop in blastocysts. Therefore, efforts should be taken in order to improve the environmental conditions and heat resistance of dairy cows in tropical and subtropical areas. Studies are been conducted to understand the molecular level by which HS disrupt reproduction aiming to develop methods to attenuate or reverse HS consequences upon fertility of dairy cows.

A baixa fertilidade de vacas Holandesas (B. taurus) repetidoras de serviço durante o estresse térmico está relacionada à sua baixa competência oocitária / The low fertility of repeat-breeder Holstein (B. taurus) cows during summer heat stress is related to a low oocyte competence

O objetivo desse estudo foi avaliar se a baixa fertilidade de vacas Holandesas repetidoras de serviço [RS; comparativamente a novilhas (NOV) e vacas próximas ao pico de lactação (PL)] está associada com comprometimento da qualidade oocitária e se esta condição é agravada pelo estresse térmico. Fêmeas das três categorias foram tratadas com o mesmo protocolo de sincronização da emergência de onda folicular. Cinco dias após o início do protocolo, a ovum pick-up (OPU) foi realizada e foram avaliados (Capítulo; Cap. 1) o número de folículos ovarianos, de oócitos totais e viáveis, temperatura retal (TR), temperatura de superfície cutânea (TC) e frequência respiratória (FR). Os oócitos viáveis foram utilizados para a produção in vitro de embriões (Cap. 2) e avaliações biomoleculares (Cap. 3). No Cap. 2, foram avaliados o desenvolvimento embrionário (taxa de clivagem, de blastocisto e de eclosão) e a qualidade dos embriões produzidos (número de células e frequência de núcleos fragmentados). No Cap. 3, realizou-se a extração de RNA e DNA de parte dos oócitos coletados para a quantificação relativa e absoluta de DNA mitocondrial (mtDNA) e a avaliação da expressão de genes relacionados à replicação/transcrição do mtDNA (PPARGC1A, NRF1, POLG, POLG2, TFAM e MT-CO1), à apoptose (BAX, BCL2 e ITM2B) e ao estresse térmico (HSP90AA1 e HSPA1AB). No Cap. 4, a taxa de concepção após IATF foi avaliada em ambas as estações do ano e nas três categorias quando o mesmo protocolo de sincronização para IATF e a mesma partida de sêmen foram utilizados. No Cap. 1, vacas RS e PL aumentaram sua FR e TR no verão em relação ao inverno (P<0,0001), enquanto as NOV mantiveram essas variáveis constantes em ambas as estações. Nas três categorias houve aumento (P<0,0001) da TC no verão, mas esta sempre foi superior (P<0,001) em vacas RS e PL do que nas NOV, independente da estação. O número de folículos e de oócitos totais e viáveis declinou nas RS e PL durante o verão, mantendo-se semelhante em NOV em ambas as estações. No Cap. 2, a taxa de clivagem foi semelhante entre categorias e estações. Já a taxa de blastocisto foi reduzida no verão nas três categorias, sendo essa queda mais acentuada nas RS. A taxa de eclosão e o número de células dos blastocistos foram menores no verão (independente de categoria). Menor número de células foi observado em embriões de RS e PL (independente da estação). Além disso, a porcentagem de núcleos fragmentados foi maior nos blastocistos das RS no verão. No Cap. 3, a expressão de ITM2B e BAX foi maior em RS durante o verão. Ainda, detectaram-se indícios da ativação de mecanismos pró-apoptóticos nos oócitos de RS (maior relação BAX/BCL-2) comparadas a PL e de mecanismos compensatórios da deficiência da função mitocondrial (menor conteúdo de mtDNA e maior expressão de PPARGCC1, NRF1, TFAM, POLG e POLG2) nas RS durante o verão em relação as demais categorias. No Cap. 4, menor taxa de concepção foi observada em RS e durante o verão. Os resultados geram evidências de que o baixo desempenho reprodutivo de vacas RS durante o verão deva estar relacionado ao comprometimento da qualidade de seus oócitos, demonstrado pelo seu reduzido conteúdo de mtDNA e elevada expressão de genes relacionados a replicação/transcrição do mtDNA, apoptose e síntese de chaperonas, culminado em baixa taxa de blastocisto e alta fragmentação nuclear destes

The aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent forThe aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent for in vitro embryo production (Chapter 2) and bimolecular evaluation (Chapter 3). In Chapter 2, embryo development (rates of cleavage, blastocyst and hatching) and quality (total number of nuclei and frequency of nuclear fragmentation) were assessed. In Chapter 3, part of the oocytes were subjected to DNA and RNA extraction to allow relative and absolute quantification of mitochondrial DNA (mtDNA), and the evaluation of the expression of genes related to mtDNA replication/transcription (PPARGC1A, NRF1, POLG, POLG2, TFAM and MT-CO1), apoptosis (BAX, BCL2 and ITM2B) and heat stress (HSP90AA1 e HSPA1AB). In Chapter 4, RB, PL and H were subjected to same protocol for fixed-time AI using the same batch of semen of a single sire in order to evaluate their P/AI during summer and winter. In Chapter 1, RB and PL had increased (P<0.0001) RR and RT during summer compared to winter; while H maintained similar RR and RT in both seasons. CT was greater (P<0.0001) during summer than winter in all categories, but it was always higher (P<0.001) in RB and PL than H, regardless of season. The numbers of follicles and total and viable oocytes were lower in RB and PL during summer than winter, and maintained stable in H in both seasons. In Chapter 2, cleavage rate was similar among categories and between seasons. However, blastocyst rate was invariably reduced during summer, but more pronouced in RB. Hatching rate and the total number of nuclei were decreased during summer, regardless of category. Lower number of nuclei was observed in RB and PL embryos compared to H, regardless of the season. Furthermore, the percentage of fragmented nuclei was greater in RB blastocysts during the summer. In Chapter 3, expressions of ITM2B and BAX were greater in RB oocytes collected during summer. Also, the activation of pro-apoptotic mechanisms (greater BAX/BCL2 ratio) was suggested in RB heat stressed-oocytes compared with PL. Activation of compensatory mechanisms of deficient mitochondrial function (low number of copies of mtDNA and increased expression of PPARGCC1, NRF1, TFAM, POLG and POLG2) were also observed in RB heat stressed-oocytes compared with PL and H. In Charpter 4, lower P/AI was observed in RB and under summer heat stress. These data provide evidences that the lower reproduction performance observed in RB during heat stress may be due to impaired oocyte quality, as shown by their reduced mtDNA content and upregulation of several genes related to mtDNA replication/transcription, apoptosis and chaperones synthesis, resulting in lower blastocyst rate and higher nuclear fragmentation of embryos

Efeito da adição de antioxidante (Trolox®) ao meio de manutenção de embriões bovinos produzidos in vivo e ao meio de transporte de oócitos bovinos aspirados de ovários provenientes de abatedouro

Os objetivos do estudo foram: avaliar o efeito antioxidante do Trolox® 1) no meio de manutenção de embriões sobre as taxas de concepção (25 e 46 dias) e a perda gestacional de receptoras bovinas Holandesas repetidoras de serviço, em dois períodos (abril-junho/setembro-novembro) e 2) no meio de transporte de oócitos bovinos aspirados de ovários de abatedouro sobre as taxas de clivagem, blastocisto e eclosão in vitro. No Experimento 1, doadoras de embriões foram superovuladas e submetidas à lavagem uterina. Os embriões colhidos foram divididos em dois grupos, deixados em meio com ou sem antioxidante, por 2 a 6h. No Experimento 2, folículos foram aspirados e os complexos cumulus-oócito grau I e II foram divididos em quatro grupos: Controle 8h, Antioxidante 8h, Controle 20h e Antioxidante 20h. Então, foram mantidos em uma transportadora de oócitos (37ºC) por 8 ou 20 horas. Após a maturação, fecundação e cultivo in vitro, as taxas de clivagem (D3), blastocisto (D6, 7 e 9) e eclosão (D11) foram avaliadas. Alíquotas de 200 ?L do meio de transporte foram retiradas em cada momento experimental (0, 8 e 20h) para realização dos testes de capacidade antioxidante total (CAT). No Experimento 1, não houve efeito de tratamento sobre as variáveis avaliadas. Foi verificado efeito de período experimental (P=0,001), categoria da doadora (P<0,05), qualidade embrionária (P=0,049) e intervalo Divisão dos Grupos-Inovulação (P<0,0001). No Experimento 2, não houve efeito de tratamento sobre as taxas analisadas. No entanto, houve redução das taxas de clivagem e blastocisto (D7 e 9) nos grupos 20 horas. Ainda, no D6 foram obtidas taxas de blastocisto semelhantes nos grupos Antioxidante 8 e 20h. A análise da CAT evidenciou que o Trolox® auxiliou o combate às ROS

The objectives of the present study were to evaluate the effect of the addition of an antioxidant (Trolox®) 1) to a holding media for bovine in vivo produced embryos, on conception rates 25 and 46 days of pregnancy and pregnancy loss, in Holstein bovine recipients at 4th or more service, during two periods of the year (April-June/September-November) and 2) to a transport media for bovine oocytes aspirated from slaughter house ovaries, on in vitro cleavage, blastocyst and hatching rates. In Experiment 1, donor cows were superovulated and submitted to uterine flushings. The recovered embryos were divided into two groups, kept in holding media with or without antioxidant, for 2-6h. In Experiment 2, follicles were aspirated and cumulus-oocyte complexes grade I and II were divided into four groups: Control 8h, Antioxidant 8h, Control 20h and Antioxidant 20h. Oocytes were kept in a transportable machine (37ºC) for 8 or 20 hours. After in vitro maturation, fertilization and culture, cleavage (D3), blastocyst (D6, 7 and 9) and hatching rates (D11) were evaluated. Samples (200 ?L) of the transport media were collected in each experimental moment (0, 8 e 20h) for total antioxidant capacity assay (TACA). In Experiment 1, no effect of treatment was observed. Effects of experimental period (P=0.001), donor category (P<0.05), embryo quality (P=0.049) and interval Group division-embryo transfer (P<0.0001) were detected. In Experiment 2, no effect of treatment was found on the analyzed rates. However, reduction on cleavage and blastocyst (D7 e 9) rates was detected on Groups 20h. Also, on D6 similar blastocyst rates were observed in Groups Antioxidant 8 and 20h. The analysis of TACA evidenced that Trolox® collaborated with the combat against the ROS