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Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.

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Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

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The objective of the present study was to evaluate the effect of estrus resynchronization (RE) after the first Timed Artificial Insemination (TAI) protocol on reproductive efficiency of suckled Bos taurus beef cows (n = 1052; Angus and Brangus) subjected to two different breeding season (BS) in Rio Grande do Sul, Brazil. In experiment 1, cows were maintained in native pasture on conventional BS (spring-summer), and in experiment 2, cows were maintained in a grazing area cultivated in an integrated crop-livestock system on modified BS (autumn -winter). Experiment 1 cows (n = 92) were randomly distributed in two reproductive managements: 1) TAI at the first day of the BS (Day 0), followed by natural service (NS) on day 10 until the end of the BS (TAI + NS; n = 45) or, 2) TAI on day 0, followed by RE 22 days after the first TAI and then followed by NS on day 42 until the end of the BS (TAI + RE22 + NS; n = 47). Experiment 2 cows (n = 960) were subjected to one of four reproductive managements: 1) only NS for 90 days of the BS (NS; n = 266); 2) TAI on day 0, followed by NS on day 10 until the end of the BS (TAI + NS; n = 200); 3) TAI on day 0, followed by RE 22 days after the first TAI and then followed by NS on day 42 until the end of the BS (TAI + RE22 + NS; n = 249) or, 4) TAI on day 0, followed by RE 30 days after the first TAI and then followed by NS on day 50 until the end of the BS (TAI + RE30 + NS; n = 245). In experiment 1, conception rate after first TAI was similar for treatments TAI + NS (42%, 19/45) and TAI + RE22 + NS (48%, 23/47; P = 0.4107). Total pregnancy rates at day 60 of the BS were 55.6% and 66.6% for TAI + NS and TAI + RE22 + NS, respectively (P = 0.006). In experiment 2, only 3% of the females submitted to NS were pregnant 30 days after the onset of BS, a lower rate compared to other groups (TAI + NS = 40%; TAI + RE22 + NS = 39.8%; TAI + RE30 + NS = 40%; P < 0.001). At 70 days of the BS, the groups TAI + RE22 + NS and TAI + RE30 + NS obtained greater pregnancy rate (66.3% and 69.4%, respectively) than other groups (NS = 16.9%; TAI + NS = 48%; P < 0.001). Pregnancy rate at the end of BS was greater in RE groups (TAI + RE22 + NS = 81.5%; TAI + RE30 + NS = 83.7%) than the NS (45.1%) or TAI + NS (71%). In conclusion, resynchronization increases pregnancy rate per AI (P/AI) and reproductive efficiency of suckled Bos taurus beef cows during spring-summer or autumn-winter breeding season in natural pasture or cultivated pasture, respectively.

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A number of reproductive biotechnologies are currently available to multiply offspring from high genetic merit animals to enhance reproductive efficiency and profitability both in dairy and beef herds. Some of these technologies such as fixed time artificial insemination (FTAI), when correctly implemented,generally allow greater reproductive performance than natural breeding. Besides the use of frozen-thawed semen during artificial insemination, cattle recipients can also be synchronized to receive embryos (produced in vivo or in vitro) at set dates with fertility results that usually outperforms natural breeding as well as artificial insemination (AI), particularly during warm seasons and in repeat breeders cows. Altogether, the use of hormonal programs to synchronize ovulation time simplify field routine, can easily fix physiological limitations related to postpartum anestrus (beef cows), poor estrus detection efficiency due to less evident estrus signs (dairy cows), making AI and ET viable to commercial herds both in terms of results and economical returns.(AU)

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A number of reproductive biotechnologies are currently available to multiply offspring from high genetic merit animals to enhance reproductive efficiency and profitability both in dairy and beef herds. Some of these technologies such as fixed time artificial insemination (FTAI), when correctly implemented,generally allow greater reproductive performance than natural breeding. Besides the use of frozen-thawed semen during artificial insemination, cattle recipients can also be synchronized to receive embryos (produced in vivo or in vitro) at set dates with fertility results that usually outperforms natural breeding as well as artificial insemination (AI), particularly during warm seasons and in repeat breeders cows. Altogether, the use of hormonal programs to synchronize ovulation time simplify field routine, can easily fix physiological limitations related to postpartum anestrus (beef cows), poor estrus detection efficiency due to less evident estrus signs (dairy cows), making AI and ET viable to commercial herds both in terms of results and economical returns.

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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)

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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].

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This study evaluated the effect of new or used P4 devices on the ovarian responses of dairy buffalo that were administered an estradiol (E2) plus progesterone (P4)-based timed artificial insemination (TAI) protocol during the breeding season. On the first day of the TAI protocol, 142 cows were randomly assigned to receive one of the following: a new device (New; 1.0 g of P4; n = 48); a device that had previously been used for 9 days (Used1x, n = 47); or a device that had previously been used for 18 days (Used2x, n = 47). Ultrasound was used to evaluate the following: the presence of a corpus luteum (CL); the diameter of the dominant follicle (ØDF) during protocol; ovulatory response; and pregnancies per AI (P/AI). Despite similar responses among the treatments, there was a significant positive association of the ØDF during TAI protocol with ovulatory responses and number of pregnancies. In conclusion, satisfactory ovarian responses and a satisfactory pregnancy rate were achieved when grazing dairy buffalo were subjected to the TAI protocol in breeding season, independent of whether a new or used P4 device was used. Furthermore, the presence of the larger follicle was associated with a higher ovulation rate and higher P/AI following TAI.

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Plasma estradiol and progesterone (P4) concentrations during the peri-ovulatory period are positively correlated with pregnancy success in cattle. The aims of this study were to assess the effects of estrus occurrence and early diestrus P4 concentrations on pregnancy per timed-embryo transfer (P/TET). A total of 267 crossbred beef heifers [222 with corpus luteum (CL) and 45 without a CL but with a follicle >8mm at beginning of the estrous synchronization protocol) received an intra-vaginal P4 device and intramuscular administration of estradiol benzoate. Progesterone devices were removed 8 days later (Day 0), and heifers received d-cloprostenol, eCG and estradiol cypionate. Estrous behavior was monitored twice daily for 3 days after P4 device removal. Plasma P4 concentration was measured by radioimmunoassay at Day 7 and Day 9. At Day 9, heifers with a CL (n=236; i.e. submission rate of 85.5%; 236/276) undergoing TET received an in vitro-produced embryo. Heifers expressing a standing behavioral estrus had a greater P/TET than heifers that did not express a standing estrus [62.4% (106/170) compared with 47.0% (31/66)]. The probability of pregnancy was positively correlated with plasma P4 concentration at TET. When heifers were grouped by quartiles of P4 concentration at TET (Q1=0.64±0.16, Q2=1.70±0.04, Q3=2.90±0.07 and Q4=5.52±0.27ng/mL) the P/TET were 45.8% (Q1; 27/59)(c), 52.25% (Q2; 31/59)(bc), 66.1% (Q3; 39/59)(ab) and 67.8% (Q4; 40/59)(a). Additionally, heifers that became pregnant had greater P4 concentrations at TET (2.87±0.16ng/mL; n=137) than heifers that did not become pregnant (2.45±0.24ng/mL; n=99). No statistical difference was observed regarding P4 concentrations on Day 7, regardless of standing estrus or pregnancy status. In cattle, manifestation of estrous behavior and plasma P4 concentration at TET increase the probability of pregnancy in in vitro-produced embryo recipients.

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O objetivo deste trabalho foi avaliar as associações entre a ocorrência de estro após os protocolos de sincronização da ovulação e diâmetro folicular no momento da inseminação artificial em tempo fixo (IATF) nas respostas ovarianas e na taxa de concepção após IATF em fêmeas zebuínas de corte. Assim, avaliaram-se as possíveis implicações práticas do uso dessas informações para a melhoria dos programas de IATF em fêmeas zebuínas lactantes. Essa tese é dividida em três capítulos. No primeiro capítulo, estudou-se a associação entre o diâmetro folicular no momento da IATF com as respostas das fêmeas após sincronização da ovulação e IATF. Observou-se que o aumento do diâmetro folicular no momento da IATF aumenta tanto a taxa de ovulação, quanto a taxa de concepção após IATF. O segundo capítulo relata a realização de quatro estudos onde foram avaliadas as associações da ocorrência de estro no intervalo entre o final do protocolo de sincronização e o momento da IATF na resposta ovariana e a taxa de concepção após a IATF. Foi observado que a ocorrência de estro após o protocolo de sincronização está associada à melhores respostas ovarianas, melhor função luteínica no ciclo subsequente e melhor taxa de concepção após IATF. Por fim, o terceiro capítulo trata da utilização das informações supracitadas para a seleção de fêmeas com maior probabilidade de gestação após IATF para receberem sêmen sexado. Os resultados são indicativos de que é possível obter maiores taxas de concepção após uso de sêmen sexado ou convencional nas fêmeas selecionadas pela ocorrência de estro ou pela presença de folículo de maior diâmetro no momento da IATF. Portanto, estratégias para melhorar a ocorrência de estro e o diâmetro folicular no momento da IATF são importantes para o sucesso dos programas de sincronização da ovulação para IATF de fêmeas zebuínas, e tais critérios podem ser utilizados na seleção de fêmeas de melhor resposta para receberem sêmen sexado

The objective of the present thesis was to evaluate the associations between the occurrence of estrus after synchronization of ovulation protocols and ovarian follicle diameter at the timed artificial insemination (TAI) on ovarian responses and conception rates after TAI in Zebu beef females. Finally, there were evaluated some practical tools using established information to select cows to receive great value semen during TAI program. This thesis was divided into three chapters. In the first chapter, it was evaluated the association between ovarian follicle diameter at the TAI moment on ovarian responses and pregnancy per TAI. It was observed that the increase in the ovarian follicle diameter at TAI increases both the ovulation and conception rates following the TAI synchronization protocol. In the second chapter, there were four studies in which was analyzed the effect of occurrence of estrus between the end of the synchronization protocol and the IATF. It was observed that the occurrence of estrus after synchronization protocol is associated with higher ovarian responses, greater luteal function on subsequent estrous cycle and higher pregnancy per TAI. Finally, the third chapter, the established information were used to select females with greater odds of pregnancy to receive sex-sorted sperm. It was possible to obtain higher conception rates following the selection of females through the occurrence of estrus or the presence of larger ovarian follicle at the time of TAI after the use either sex-sorted or non sex-sorted sperm. Therefore strategies to improve the occurrence of estrus and the ovarian follicle diameter at TAI moment are important to improve the reproductive outcomes following synchronization of ovulation protocols for TAI in lactating zebu cows. Besides, such selection criteria can be used to select females with greater odds of pregnancy to receive sex-sorted sperm

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