Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have causes other than pregnancy loss. We enrolled Holstein dairy cows ≥50 DIM from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an AI or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a corpus luteum (CL) on d 17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, BCS, and the presence of a CL at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% Bayesian credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92; 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.

Evaluation of pregnancy per artificial insemination of dairy animals detected in estrus during the synchronization protocol and assigned to conventional or sexed semen.

Three experiments were designed to evaluate the effects of conventional and sexed-semen on reproductive performance of dairy females detected in estrus. In Exp. 1, 978 lactating Holstein cows and 116 Holstein heifers were used. Cows or heifers were randomly assigned to receive conventional (CONV) or sexed (SEXD) semen on day 0 of a TAI protocol. The TAI was performed using commercial CONV or SEXD frozen-thawed semen from the same 6 Holstein bulls, and SEXD semen was sexed through fluorescence cytometry. In Exp. 2, 390 lactating Holstein × Gir dairy cows and 344 Holstein × Gir heifers were used. Cows and heifers were randomly assigned to receive CONV or SEXD semen on day 0 with frozen-thawed semen from 5 Holstein × Gir bulls. The SEXD was sorted through the Sexcel™ methodology. In Exp. 3, 789 primiparous lactating Holstein cows were enrolled. Cows were randomly assigned to receive CONV or SEXD semen from 5 Holstein bulls, and the SEXD semen was sexed using the same semen allocation methodology described in Exp. 2. All data were analyzed using SAS. Regardless of the experiment, no treatment × bull interaction was observed for any of the variables analyzed herein (P ≥ 0.20). In Exp. 1, multiparous cows assigned to CONV had a greater P/AI vs. cohorts assigned to SEXD (P < 0.01) and a similar response tended to be observed for secundiparous cows (P = 0.10). No treatment effects were further observed when the effects of semen on day 60 P/AI were stratified by heat stress, milk yield, and CL at the beginning of the protocol. In Exp. 2, no P/AI differences between treatment were detected either on days 32 (P = 0.32) or 60 of pregnancy (P = 0.20). In Exp. 3, cows assigned to SEXD had reduced P/AI on day 32 (P = 0.03) and tended to have a reduced P/AI on day 60 of pregnancy (P = 0.06). No further treatment effects or interactions were observed herein (P ≥ 0.13). Overall P/AI of SEXD semen was 78% of the CONV (P < 0.01), but the variation of these results requires some attention. Moreover, an additional analysis was performed considering the median value of P/AI in bulls assigned to CONV and SEXD, and bulls that had a greater P/AI in CONV also had a greater P/AI in SEXD (P = 0.03). In summary, although not directly comparable, the sexing technologies herein provided similar results in heifers, but not in other categories (primiparous, secundiparous, and multiparous cows). Additionally, the P/AI of a sexed semen is associated to its performance on the conventional semen counterpart. Moreover, in the present experiment, no other parameters were shown to impact fertility of dairy females assigned to conventional or sexed-semen.

Effects of additional gonadotropin-releasing hormone and prostaglandin F2α treatment to an estradiol/progesterone-based embryo transfer protocol for recipient lactating dairy cows.

This study was designed to evaluate whether the utilization of a second PGF2α treatment at the end of an estradiol/progesterone (E2/P4)-based protocol with or without GnRH at the beginning of the protocol would improve pregnancy rates of lactating Holstein cows assigned to timed embryo transfer. A total of 501 lactating Holstein cows in 5 farms were enrolled in the experiment. Within farm, cows were blocked by parity and, within block, were assigned randomly to (1) insertion of an intravaginal P4 device (controlled internal drug-releasing device; CIDR) and estradiol benzoate on d -11, PGF2α on d -4, CIDR withdrawal and an injection of estradiol cypionate on d -2, and timed embryo transfer on d 7 (1-PGF; n = 164); (2) the same treatments as 1-PGF, but with PGF2α administered on d -4 and -2 (2-PGF; n = 171); and (3) 2-PGF with the addition of a GnRH treatment on d -11 (GnRH+2-PGF; n = 166). Ovaries were scanned by transrectal ultrasonography on d -11, -4, and 7, and blood samples were collected on d -11, -4, 0, and 7 for P4 determination. Treatment comparisons were performed using contrasts. The proportion of cows with a new corpus luteum on d -4 was greater in GnRH+2-PGF cows. Cows in 1-PGF had a greater P4 concentration on d 0 but lesser P4 on d 7 compared with cows in the other groups. Cows assigned to receive 2-PGF (2-PGF and GnRH+2-PGF) had greater estrus expression, and a greater proportion of cows ovulated to estradiol cypionate. No further contrast effects were observed for follicle diameter, double ovulation rate, pregnancy per embryo transfer (P/ET) on d 32 and 60, or pregnancy loss. As P4 concentration on d -4 increased, P/ET on d 60 tended to increase. Cows with P4 ≥3.66 ng/mL on d -4 had greater P/ET on d 32 and 60 than those with P4 below that threshold. Regardless of treatment, cows with P4 concentration ≥3.66 ng/mL also had a greater pregnancy per synchronized protocol (P/SP) on d 60. Also, a P4 concentration on d -4 (low or high) × follicle diameter (continuous) interaction tendency was observed when evaluating P/ET. Although P/ET did not differ among cows with different follicles sizes with reduced P4 concentration on d -4 (<3.66 ng/mL), it increased in cows with larger follicles exposed to increased P4 concentration (≥3.66 ng/mL). When P4 on d 0 was evaluated, P/ET on d 32 and 60 was greater for cows with low (≤0.09 ng/mL) versus high (>0.21 ng/mL) P4; as P4 concentration on d 0 increased, P/ET linearly decreased. In summary, cows with increased P4 concentrations during growth of the ovulatory follicular wave had improved P/ET. Administering a second PGF2α dose reduced P4 concentration on d 0 and increased ovulatory response to the protocol, but no benefits were observed on P/ET or P/SP.

Effects of estradiol cypionate dose as an ovulatory stimulus on reproductive performance of lactating dairy cows during the summer season.

This experiment was designed to evaluate the effects of increasing doses of estradiol cypionate (ECP) on reproductive function of lactating dairy cows during the summer. In Exp. 1, 643 lactating Holstein cows were blocked by parity and assigned to receive 1) an intravaginal P4 device (1.9 g of P4) and 2.0 mg of estradiol benzoate on day -11, 25 mg (i.m.) of dinoprost tromethamine on day -4, 1.0 mg (i.m.) of estradiol cypionate and CIDR withdrawal on day -2, followed by TAI on day 0 (n = 326; ECP-1) or 2) the same synchronization protocol with 2.0 mg of ECP on day -2 (n = 317; ECP-2). In both treatments, cows were TAI on day 0 of the protocol, and cow rectal temperature was measured on days -2, 0, and 7. In Exp. 2, 608 lactating crossbred Holstein × Gir dairy cows were blocked by parity and enrolled to the same treatments as in Exp. 1, but on day 7, cows received one viable embryo into the uterine horn. In Exp. 1, a greater percentage of ECP-2 cows were detected on estrus (81.3 vs. 91.1%, respectively). A treatment × body condition score (BCS) interaction was observed on day 60 pregnancy per AI (P/AI), as ECP-2 cows with a BCS <2.75 had a greater P/AI vs. ECP-1, but an opposite result was observed in cows with a BCS ≥2.75. Regardless of treatment, there were effects of mean rectal temperature and heat stress events on P/AI. Treatment affected the diameter of the ovulatory follicle at TAI (ECP-1 = 15.3 mm vs. ECP-2 = 14.8 mm) and a greater percentage of ECP-1 cows had larger follicles (≥16.5 mm), but ECP-2 resulted in a greater incidence of early ovulatory cows (ovulating before day 0). Therefore, follicle diameter at TAI affected P/AI on day 60 in cows receiving ECP-2 and tended to affect ECP-1 cows. A treatment effect was observed on time to estrus following ECP treatments and a reduced proportion of ECP-2 cows showed estrus at TAI. Regardless of treatment, cows detected on estrus 48 h after ECP administration had a greater P/AI on day 60 vs. cows detected on estrus 24 h. In Exp. 2, a greater percentage of ECP-2 cows were detected on estrus (68.3 vs. 81.4%, respectively). In summary, a greater dose of ECP increased the percentage of animals expressing estrus, but it did not benefit the reproductive function of lactating dairy cows during the summer, regardless if animals were assigned to a TAI or timed-embryo transfer (TET) protocol.

Effect of salivary CYP4EM1 and CYP4EM2 gene silencing on the life span of Chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae) exposed to sublethal dose of deltamethrin.

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas.

Increasing the length of an estradiol with progesterone timed artificial insemination protocol with 2 controlled internal drug release devices improves pregnancy per artificial insemination in lactating dairy cows.

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day -11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d -4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d -2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d -12; 25 mg of PG and withdrawal of 1 CIDR on d -4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d -2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13-15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone-based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows.

Evaluation of presynchronization and addition of GnRH at the beginning of an estradiol/progesterone protocol on circulating progesterone and fertility of lactating dairy cows.

The objectives of this study were to determine if the utilization of a presynchronization strategy would improve fertility at first artificial insemination (AI) during an E2/P4 ovulation synchronization protocol with or without GnRH administration at the beginning of the protocol. This experiment was conducted using cows (n = 665) at their first postpartum service and the following breeding treatment: CIDR insertion and 2.0 mg of estradiol benzoate (EB) on day -11; 25 mg dinoprost tromethamine (PG) on day -4; PG, CIDR withdrawal, and 1.0 mg of estradiol cypionate (ECP) on day -2; timed-AI on day 0. At 31 ± 3 days postpartum, cows were randomly allocated to one of three treatments on a weekly basis: 1) -P + GnRH: cows assigned to the breeding protocol with 100 µg of GnRH on day -11, 2) P + GnRH: cows assigned to a presynchronization protocol using CIDR insertion +2 mg EB on day -28, PG + ECP and CIDR withdrawal on day -21, and beginning of the breeding protocol plus GnRH (100 µg) on day -11, and 3) +P-GnRH: cows assigned to a presynchronization protocol and the breeding treatment without GnRH on day -11. No treatment effects were observed on P/AI at the pregnancy diagnoses on days 32 and 60, or for pregnancy losses between days 32 and 60 of pregnancy whether analyses included all cows or only cows that ovulated near TAI. Moreover, milk yield negatively affected P/AI. Cows with greater circulating P4 concentrations on day -4 had greater P/AI on day 60. Cows without CL on day -11 had a reduced P/AI and this effect was more significant in cows not treated with GnRH. Cows assigned to -P + GnRH had the lowest circulating P4 concentration on day -4 (3.4 ± 0.16 ng/mL), followed by + P-GnRH (4.56 ± 0.17 ng/mL), and +P + GnRH (5.08 ± 0.17 ng/mL) cohorts. The data of the current study suggest that the combination of a Presynch and GnRH administration at the beginning of a TAI protocol was the most effective way to increase the % of cows with a functional CL and with elevated circulating P4 concentrations at the time of PG treatment.

Somatic cell count and type of intramammary infection impacts fertility from in vitro produced embryo transfer.

The objective of this study was to assess the impact of mastitis-causing bacteria and somatic cell count (SCC) on pregnancy per embryo transfer (P/ET) in Holstein-Gir crossbred (Girolando) lactating dairy cows. Cows (n = 1397) were subjected to a timed-embryo transfer protocol. Milk samples were collected two days before embryo transfer for SCC and bacteriological culture analyses. Pregnancy diagnosis was performed on days 31 and 66 after timed-embryo transfer. The animals were grouped according to the National Mastitis Council recommendations: Gram-positive environmental (EV+), Gram-negative environmental (EV-), Gram-positive contagious (C+), coagulase-negative staphylococci (CNS) and control (no bacterial growth). Additional analysis was made by categorizing bacteria based on degree of pathogenicity (Major or Minor). Bacterial growth reduced P/ET (P < .01) at both 31 and 66 days of gestation. The P/ET was lower (P < .05) at 31 days in EV- (30.1%) and EV+ (29.9%) groups and tended (P = .09) to be lower in the C+ group (36.6%) than the control group (44.0%). The P/ET from the Major group at 31 days of gestation was lower (P = .03) compared with the Minor and control groups (32.1 vs 41.1 vs 43.2%, respectively). Cows with SCC > 400,000 cells/mL had lower P/ET (P < .01) than animals with SCC < 200,000 cells/mL at both 31 (30.4% vs 40.8%) and 66 days (24.7% vs 32.2%) of gestation. Pregnancy loss was not different between bacterial isolates and SCC categories. Elevated SCC significantly reduced P/ET, whereas environmental agents and those with Major pathogenicity yielded the greatest reduction in P/ET.

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d -11, PGF2α on d -4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF2α) on d -2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d -11, 1 CIDR withdrawn + PGF2α on d -4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF2α on d -2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d -11 or d -4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF2α treatment (d -4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.

Comparison of fertility following use of one versus two intravaginal progesterone inserts in dairy cows without a CL during a synchronization protocol before timed AI or timed embryo transfer.

The objective was to evaluate the effect of increased progesterone (P4) during preovulatory follicle growth during timed AI (TAI) or timed embryo transfer (TET) protocols. Lactating dairy cows with no CL and low circulating P4 (≤1.0 ng/mL) were submitted to a protocol using one or two intravaginal P4 implants (controlled intravaginal releasing device [CIDRs]), and were bred to TAI or TET. The low P4 cows for this experiment were identified on nine farms, four utilized TAI (n = 326 of 1160 cows examined), and five utilized TET (n = 445 of 1396). All cows were synchronized by insertion of P4 implant(s) (CIDR[s]) at start of protocol (Day -11) and simultaneous treatment with 2 mg of E2-benzoate. After 7 days, cows were treated with PGF (Day -4) and 2 days later treated with 1.0-mg E2-cypionate and CIDR(s) were removed (Day -2). Cows received TAI on Day 0 or TET on Day 7. Cows were randomly assigned to receive either one or two CIDRs on Day -11 until Day -2 (1CIDR vs. 2CIDR). Presence of CL was determined by ultrasound on Day -11 and Day 7 after protocol (to determine ovulation to protocol), P4 concentrations were determined on a subset of cows (Day -11, Day -4, Day 7), and ovulatory follicle diameter was evaluated on Day 0. Pregnancy success (P/AI or P/ET) was evaluated on Days 32 and 60. The 2CIDR treatment increased circulating P4 by Day -4 (1.77 ± 0.23 vs. 2.18 ± 0.24 ng/mL) but had no effect on ovulation at the end of protocol (83.6 vs. 82.6%) or ovulatory follicle diameter (15.6 ± 0.3 vs. 15.3 ± 0.3 mm). If only cows that ovulated to the protocol were included, 1CIDR tended to have lower P/AI than 2CIDR by Day 32 (42.8 vs. 52.6%; P = 0.10) and Day 60 (37.7 vs. 48.1%; P = 0.08) but there was no effect on pregnancy loss. There was an interaction (P = 0.05) between ovulatory follicle diameter and CIDR treatment on P/AI (Day 60). In cows ovulating larger follicles (≥14 mm), 2CIDR treatment increased P/AI compared with 1CIDR (53.3 vs. 34.9%; P = 0.02) but not in cows ovulating small follicles (<14 mm). There was no effect of treatment on P/ET on Day 32 (30.0% vs. 32.0%) or Day 60 (24.7% vs. 25.6%). Thus, these results add evidence to the concept that increased circulating P4 during preovulatory follicle development may improve P/AI, most likely due to improved oocyte quality in cows that ovulate larger follicles, since improvement was only in cows ovulating larger follicles and no effect of preovulatory P4 was observed in cows that received ET.

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.(AU)

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.

Expression of estrus improves fertility and decreases pregnancy losses in lactating dairy cows that receive artificial insemination or embryo transfer.

The objective was to evaluate if expression of estrus by dairy cattle altered fertility in timed artificial insemination (AI; n=5,430) or timed embryo transfer (ET; n=2,003) programs that used estradiol and progesterone (P4) to synchronize ovulation. Ovarian ultrasonography was performed on d 0 (time of AI) and 7 to determine ovulatory follicle diameter and ovulation. Only cows with a visible corpus luteum on d 7 were used in this study. At the time of controlled internal drug release removal, all cows received a tail-head device for detection of estrus and were considered in estrus when the paint of the device was completely removed by d 0. Circulating P4 concentrations were evaluated on d 7. Pregnancies per AI (P/AI) or ET (P/ET) were determined by ultrasonography on d 32 and 60. At d-32 pregnancy diagnosis, cows with expression of estrus had increased P/AI [no estrus=25.5% (222/846) vs. estrus=38.9% (1,785/4,584)] and P/ET [no estrus=32.7% (193/606) vs. estrus=46.2% (645/1,397)]. Similarly, at d-60 pregnancy diagnosis, expression of estrus increased P/AI [no estrus=20.1% (179/846) vs. estrus=33.3% (1,530/4,584)] and P/ET [no estrus=25.1% (150/606) vs. estrus=37.5% (525/1,397)]. Pregnancy loss was lower in cows that expressed estrus in timed AI [TAI; no estrus=20.1% (43/222) vs. estrus 14.4% (255/1,785)] and timed ET [TET; no estrus=22.7% (43/193) vs. estrus=18.6% (120/645)] compared with cows with no estrus. Independent of expression of estrus cows ovulating either too small or too large of follicles had lower P/AI. No effect of ovulatory follicle diameter on P/ET was noted in cows that expressed estrus; although, cows that did not express estrus tended to have lower P/ET if they ovulated larger follicles. In cows that showed estrus, follicle diameter did not affect pregnancy loss, but cows that did not show estrus and ovulated larger follicles tended to have greater pregnancy loss after TAI and had greater pregnancy loss on TET. A positive effect of d-7 P4 concentrations on P/AI was observed, independent of estrus. In contrast, no effect of P4 was found on d 7 on P/ET. Thus, expression of estrus during protocols for TAI or TET is associated with an increase in fertility and reduction in pregnancy loss. During TAI programs, optimizing follicle diameter and increasing circulating P4 on d 7 after AI were also associated with increased fertility, independent of expression of estrus. However, in cows with TET, the association of fertility with either ovulatory follicle diameter or P4 on d 7 was less dramatic and seemed to be related to whether cows expressed estrus.

Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.

Our hypothesis was that fertility could be increased in a timed artificial insemination (TAI) protocol based on estradiol (E2) and progesterone (P4) by combining GnRH with E2-benzoate at the start of the protocol to increase circulating P4 during preovulatory follicle development and by using 2 prostaglandin F2α (PGF) treatments at the end to decrease P4 near TAI. Lactating Holstein cows (n=1,808) were randomly assigned during the cool or hot season of the year to receive TAI (d 0) following 1 of 3 treatments: (1) control: controlled internal drug-release insert + 2mg of E2-benzoate on d -11, PGF on d -4, controlled internal drug-release insert withdrawal + 1.0mg of E2-cypionate on d -2, and TAI on d 0; (2) 2PGF: identical to control protocol with addition of a second PGF treatment on d -2; (3) GnRH: identical to 2PGF protocol with addition of a 100-µg GnRH treatment on d -11. Pregnancy diagnoses were performed on d 32 and 60 after TAI. Season had major effects on many reproductive measures, with cool season greater than hot season in percentage of cows with corpus luteum (CL) at PGF (62.9 vs. 56.2%), ovulatory follicle diameter (15.7 vs. 14.8mm), expression of estrus (86.7 vs. 79.9%), ovulation following the protocol (89.7 vs. 84.3%), and pregnancies per artificial insemination (P/AI; 45.4 vs. 21.4%). The GnRH protocol increased percentage of cows with CL (control=56.9%; 2PGF=55.8%; GnRH=70.5%) and P4 at PGF (control=3.28±0.22; 2PGF=3.35±0.22; GnRH=3.70±0.21ng/mL), compared with control and 2PGF protocols. The GnRH protocol increased P/AI at the pregnancy diagnosis at 32d [37.3% (219/595)] and 60d [31% (179/595)] after TAI, compared with control [30.0% (177/604); 25.1% (145/604)], with intermediate results with 2PGF protocol [33.2% (196/609); 28.0% (164/609)]. The positive effects of GnRH treatment on P/AI were only detected during the cool season (GnRH=50.9%; 2PGF=44.2%; control=41.0%) and not during the hot season. In addition, the effect of GnRH was only observed in cows with low P4 (<3ng/mL) at the start of the protocol and not in cows that began the protocol with high P4. Furthermore, presence of CL at PGF interacted with follicle diameter such that cows with a CL at PGF had greater P/AI if they ovulated larger rather than smaller follicles near TAI. Thus, fertility to TAI can be improved by inducing ovulation at the beginning of an E2/P4-based protocol using GnRH treatment, particularly during the cool season of the year and in cows with low P4 at the start of the protocol.

Effect of interval from induction of puberty to initiation of a timed AI protocol on pregnancy rate in Nellore heifers.

Prepubertal Bos indicus heifers (n = 774) were submitted to an E2/P4-based timed artificial insemination (TAI) protocol at three different intervals after induction of their pubertal ovulation by insertion of an intravaginal progesterone (P4) device for 12 days. Heifers were randomly assigned to start the TAI protocol at 10 (group 10; n = 253), 12 (group 12; n = 265), or 14 (group 14; n = 256) days after the P4 device was removed. The TAI protocol consisted of the following: insertion of intravaginal device containing P4 (Controlled internal drug release [CIDR]; previously used twice for 9 days each) + estradiol benzoate (2 mg) on Day 0, CIDR withdrawal + estradiol cypionate (0.5 mg) and PGF2α (12.5 mg) on Day 9, and TAI on Day 11. A subgroup of heifers (n = 472) was evaluated by ultrasound on Days 9 and 11 to evaluate the ovaries and to determine P4 concentrations on Day 9. On Day 9, more (P < 0.05) CLs were present, and follicular diameter was smaller (P < 0.05) for group 10 than for groups 12 and 14 (38.4%, 29.3%, and 23.3% with CL and 9.4 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.1 mm diameter, respectively), but P4 concentrations did not differ (P > 0.1) between treatments (2.4 ± 0.06 ng/mL). Follicular diameter at TAI (11.08 ± 0.09 mm) and ovulation rate (88.4%) did not differ between treatments (P > 0.1). However, conception and pregnancy rates for all heifers were greater (P < 0.05) in group 12 (50.4% and 45.5%, respectively) than in group 10 (38.2% and 33.7%, respectively), with group 14 intermediate to other treatments (45.6% and 40.6%, respectively). The final pregnancy rate did not differ between treatments (80.9%). In conclusion, a 12-day interval from the end of the puberty induction protocol to the start of the TAI protocol resulted in greater conception and pregnancy rates in prepubertal Nellore heifers.

Increasing length of an estradiol and progesterone timed artificial insemination protocol decreases pregnancy losses in lactating dairy cows.

Our hypothesis was that increasing the length of an estradiol and progesterone (P4) timed artificial insemination (TAI) protocol would improve pregnancy per artificial insemination (P/AI). Lactating Holstein cows (n=759) yielding 31 ± 0.30 kg of milk/d with a detectable corpus luteum (CL) at d -11 were randomly assigned to receive TAI (d 0) following 1 of 2 treatments: (8d) d -10 = controlled internal drug release (CIDR) and 2.0mg of estradiol benzoate, d -3 = PGF2α(25mg of dinoprost tromethamine), d -2 = CIDR removal and 1.0mg of estradiol cypionate, d 0 = TAI; or (9 d) d -11 = CIDR and estradiol benzoate, d -4 = PGF2α, d -2 CIDR removal and estradiol cypionate, d 0 TAI. Cows were considered to have their estrous cycle synchronized in response to the protocol by the absence of a CL at artificial insemination (d 0) and presence of a CL on d 7. Pregnancy diagnoses were performed on d 32 and 60. The ovulatory follicle diameter at TAI (d 0) did not differ between treatments (14.7 ± 0.39 vs. 15.0 ± 0.40 mm for 8 and 9 d, respectively). The 9 d cows tended to have greater P4 concentrations on d 7 in synchronized cows (3.14 ± 0.18 ng/mL) than the 8d cows (3.05 ± 0.18 ng/mL). Although the P/AI at d 32 [45 (175/385) vs. 43.9% (166/374) for 8d and 9 d, respectively] and 60 [38.1 (150/385) vs. 40.4% (154/374) for 8d and 9 d, respectively] was not different, the 9 d cows had lower pregnancy losses [7.6% (12/166)] than 8d cows [14.7% (25/175)]. The cows in the 9 d program were more likely to be detected in estrus [72.0% (269/374)] compared with 8d cows [62% (240/385)]. Expression of estrus improved synchronization [97.4 (489/501) vs. 81% (202/248)], P4 concentrations at d 7 (3.22 ± 0.16 vs. 2.77 ± 0.17 ng/mL), P/AI at d 32 [51.2 (252/489) vs. 39.4% (81/202)], P/AI at d 60 [46.3 (230/489) vs. 31.1% (66/202)], and decreased pregnancy loss [9.3 (22/252) vs. 19.8% (15/81)] compared with cows that did not show estrus, respectively. Cows not detected in estrus with small (<11 mm) or large follicles (>17 mm) had greater pregnancy loss; however, in cows detected in estrus, no effect of follicle diameter on pregnancy loss was observed. In conclusion, increasing the length of the protocol for TAI increased the percentage of cows detected in estrus and decreased pregnancy loss.

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

Two experiments were designed to evaluate the effects of treatments with low versus high serum progesterone (P4) concentrations on factors associated with pregnancy success in postpubertal Nellore heifers submitted to either conventional or fixed timed artificial insemination (FTAI). Heifers were synchronized with a new controlled internal drug release device (CIDR; 1.9 g of P4 [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus 2 mg of estradiol (E2) benzoate on Day 0 and 12.5 mg of prostaglandin F2α on Day 7. In experiment 1 (n = 723), CIDR were removed on Day 7 or 9 and heifers were inseminated after estrus detection. In experiment 2 (n = 1083), CIDR were all removed on Day 9 and FTAI was performed either 48 hours later in heifers that received E2 cypionate (ECP) on Day 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH (100 µg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P4 concentrations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment 1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day 7, 3.76 ± 0.08 days vs. Day 9, 2.90 ± 0.07; P < 0.01) and improved conception (Day 7, 57.1% vs. Day 9, 65.8%; P = 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P = 0.04). In experiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus (E48, 40.9%(a); G54, 17.1%(c); and G72, 32.0%(b)), but the pregnancy rate was not affected (P = 0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3 increased follicle diameter at FTAI (CIDR1, 11.07 ± 0.10 vs. CIDR3, 11.61 ± 0.10 mm; P < 0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception (CIDR1, 42.2 vs. CIDR3, 45.1%; P = 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P = 0.11). In conclusion, length of treatment with P4 affected the fertility of heifers bred based on estrus detection. When the heifers were submitted to FTAI protocol, follicle diameter at FTAI (≤10.7 mm, 23.6%; 10.8-15.7 mm, 51.5%; ≥15.8 mm, 30.0%; P < 0.01) was the main factor that affected conception and pregnancy rates.

Timed artificial insemination programs during the summer in lactating dairy cows: comparison of the 5-d Cosynch protocol with an estrogen/progesterone-based protocol.

The objective of this study was to compare a GnRH-based to an estrogen/progesterone (E2/P4)-based protocol for estrous cycle synchronization and fixed timed artificial insemination (TAI), both designed for synchronization of ovulation and to reduce the period from follicular emergence until ovulation in cows with a synchronized follicular wave. A total of 1,190 lactating Holstein cows (primiparous: n=685 and multiparous: n=505) yielding 26.5 ± 0.30 kg of milk/d at 177 ± 5.02 d in milk were randomly assigned to receive one of the following programs: 5-d Cosynch protocol [d -8: controlled internal drug release (CIDR) + GnRH; d -3: CIDR removal + PGF2α; d -2: PGF2α; d 0: TAI + GnRH] or E2/P4 protocol (d -10: CIDR + estradiol benzoate; d -3: PGF2α; d -2: CIDR removal + estradiol cypionate; d 0: TAI). Rectal temperature and circulating progesterone (P4) were measured on d -3, -2, 0 (TAI), and 7. The estrous cycle was considered to be synchronized when P4 was ≥ 1.0 ng/mL on d 7 in cows that had luteolysis (P4 ≤ 0.4 ng/mL on d 0). To evaluate the effects of heat stress, cows were classified by number of heat stress events: 0, 1, and 2-or-more measurements of elevated body temperature (≥ 39.1 °C). Pregnancy success (pregnancy per artificial insemination, P/AI) was determined at d 32 and 60 after TAI. The cows in the 5-d Cosynch protocol had increased circulating P4 at the time of PGF2α injection (2.66 ± 0.13 vs. 1.66 ± 0.13 ng/mL). The cows in the E2/P4 protocol were more likely to be detected in estrus (62.8 vs. 43.4%) compared with the cows in the 5-d Cosynch protocol, and expression of estrus improved P/AI in both treatments. The cows in the 5-d Cosynch protocol had greater percentage of synchronized estrous cycle (78.2%), compared with cows in the E2/P4 protocol (70.7%). On d 60, the E2/P4 protocol tended to improve P/AI (20.7 vs. 16.7%) and reduced pregnancy loss from 32 to 60 d (11.0 vs. 19.6%), compared with the 5-d Cosynch protocol. In cows withtheir estrous cycle synchronized, the E2/P4 protocol had greater P/AI (25.6 vs. 17.7%) on d 60 and lower pregnancy loss from 32 to 60 d (6.7 vs. 21.7%) compared with cows in the 5-d Cosynch protocol. Follicle diameter affected pregnancy loss from 32 to 60d only in the cows in the 5-d Cosynch protocol, with smaller follicles resulting in greater pregnancy loss. Pregnancy per AI at d 60 was different between protocols in the cows with 2 or more measurements of heat stress (5-d Cosynch=12.2% vs. E2/P4=22.8%), but not in the cows without or with 1 heat stress measurement. In conclusion, the 5-d Cosynch protocol apparently produced better estrous cycle synchronization than the E2/P4 protocol but did not improve P/AI. The potential explanation for these results is that increased E2 concentrations during the periovulatory period can improve pregnancy success and pregnancy maintenance, and this effect appears to be greatest in heat-stressed cows when circulating E2 may be reduced.

Timing of prostaglandin F2α treatment in an estrogen-based protocol for timed artificial insemination or timed embryo transfer in lactating dairy cows.

Objectives were to investigate progesterone concentrations and fertility comparing 2 different intervals from PGF(2α) treatment and induced ovulation in an estrogen-based ovulation synchronization protocol for timed artificial insemination (TAI) or timed embryo transfer (TET) in lactating dairy cows. A total of 1,058 lactating Holstein cows [primiparous (n=371) and multiparous (n=687)], yielding 34.1 ± 0.33 kg of milk/d at various days in milk were randomly assigned to receive treatment with PGF(2α) on either d 7 or 8 of the following protocol: d 0: 2mg of estradiol benzoate + controlled internal drug release device; d 8: controlled internal drug release device removal + 1.0mg of estradiol cypionate; d 10: TAI or d 17: TET. Only cows with a corpus luteum at d 17 received an embryo and all cows received GnRH at TET. Pregnancy diagnoses were performed by detection (transrectal ultrasonography) of an embryo on d 28 or a fetus on d 60. Fertility [pregnancy per artificial insemination (P/AI) or pregnancy per embryo transfer (P/ET)] was affected by breeding technique (AI vs. ET) and time of PGF(2α) treatment (d 7 vs. 8) at the 28-d pregnancy diagnosis for TAI [32.9% (238) vs. 20.6% (168)] and TET cows [47% (243) vs. 40.7% (244)] and at the 60-d pregnancy diagnosis for TAI [30% (238) vs. 19.2% (168)] and TET cows [37.9% (243) vs. 33.5% (244)]. The progesterone (P4) concentration at d 10 altered fertility in TAI cows, with higher P/AI in cows with P4 concentration <0.1 ng/mL compared with cows with P4 concentration ≥ 0.1 ng/mL, and in ET cows, with higher P/ET in cows with P4 concentration <0.22 ng/mL compared with cows with P4 concentration ≥ 0.22 ng/mL. Prostaglandin F(2α) treatment at d 7 increased the percentage of cows with P4 <0.1 ng/mL on d 10 [39.4 (85) vs. 23.2 (54)]. Reducing the period between PGF(2α) and TAI from 72 to 48 h in dairy cows resulted in a clear reduction in fertility in cows bred by TAI and a subtle negative effect in cows that received TET. The earlier PGF(2α) treatment benefits are most likely mediated through gamete transport, fertilization, or early embryo development and a more subtle effect of earlier PGF(2α) treatment that may be mediated through changes in the uterine or hormonal environment that manifests itself after ET on d 7.

Effects of vaccination against reproductive diseases on reproductive performance of lactating dairy cows submitted to AI.

Four experiments evaluated the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp. on reproductive performance of lactating dairy cows without (experiments 1, 2, and 3) or with previous vaccination against these diseases (experiment 4). Cows were assigned to a fixed-time AI protocol (FTAI; d -11 to 0) in all experiments, as well as AI 12 h upon estrus detection in experiment 3. Pregnancy status was determined with transrectal ultrasonography on d 30 and 71 (d 60 for experiment 3) after AI. Pregnancy loss was considered in cows pregnant on d 30 but non-pregnant on the subsequent evaluation. In experiment 1, 853 cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI (d -11) and 30 d after AI. Pregnancy loss was reduced (P = 0.03) in VAC cows compared with CON. In experiment 2, 287 cows received VAC or CON 30 d prior to (d -41) and at the beginning (d -11) of the FTAI. Pregnancy rates on d 30 and 71 were greater (P ≤ 0.03) in VAC cows compared with CON. In experiment 3, 1680 cows with more than 28 d in milk were randomly assigned to receive VAC or CON with doses administered 14 d apart, and inseminated within 15-135 d after the second dose. Pregnancy rates on d 30 and 60 were greater (P ≤ 0.02) in VAC cows compared with CON. In experiment 4, 820 cows received (REVAC) or not (CON) revaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI protocol (d -11). Pregnancy rates and loss were similar (P ≥ 0.54) between treatments. Hence, vaccinating naïve cows against BoHV-1, BVDV, and Leptospira spp. improved reproductive efficiency in dairy production systems, particularly when both doses were administered prior to AI.