Abstract

The objectives were to evaluate the effects of recipient category, season and administration of a gonadorelin analogue at the time of fixed-time embryo transfer (FTET) on pregnancy per FTET at 30 and 60 days after oestrus and on pregnancy losses (PL). Recipients were randomly assigned to: treated group (n = 624), in which recipients received an intramuscular injection of 0.2 mg of gonadorelin (Fertagyl®) at FTET; or a control group (n = 687) that remained untreated. Recipients were previously treated with a synchronisation protocol based on progesterone and oestradiol. All embryos were produced in vitro. The data with binomial distribution were analysed by multivariate logistic regression, using the GLIMIX procedure of SAS. Higher P/FTET was observed at 30 days (45.8 vs. 40.0%; P = 0.03) and 60 days (43.0 vs. 37.0%; P = 0.01) in the treated group. There was a tendency toward reduced PL in the treated group (4.0 vs. 7.0%; P = 0.09). Dry cows (2.70%) and lactating cows (2.47%) had less PL (P = 0.001) compared with heifers (10.42%). In the spring/summer season the P/ FTET at 60 days was smaller (P =0.024). Greater PL tended to occur in the warmer season. Treatment with gonadorelin at the time of bovine ET increased the pregnancy per ET at days 30 and 60 and reduced PL. Additionally, dry and lactating recipient cows showed a lower PL rate compared to heifers. Furthermore, ET performed in the warmer seasons of the year resulted in a lower pregnancy rate at day 60 and greater PL.

Keywords:
reproductive efficiency; dairy cattle; reproduction

Resumo

Objetivou-se avaliar os efeitos da administração de análogo da gonadorelina no momento da transferência de embrião em tempo fixo (TETF), da categoria da receptora e da estação do ano na prenhez por TETF aos 30 (P/TETF 30) e 60 (P/ TETF60) após o estro e nas perdas gestacionais (PG). As receptoras foram distribuídas aleatoriamente: grupo tratado (n = 624), onde as receptoras receberam injeção intramuscular de 0,2 mg de gonadorelina (Fertagyl®) na TETF e grupo controle (n =687) permaneceu sem tratamento. As receptoras foram previamente tratadas com protocolo de sincronização à base de progesterona e estradiol. Todos os embriões foram produzidos in vitro. Os dados foram analisados por regressão logística multivariada, utilizando o procedimento GLIMIX do SAS. Foi detectada maior P/TETF30 (45,8 vs. 40,0%; P =0,03) e P/TETF60 (43,0 vs. 37,0%; P =0,01) no grupo tratado. Houve tendência de redução da PG no grupo tratado (4,0 vs. 7,0%; P =0,09). Vacas secas (2,70%) e vacas em lactação (2,47%) apresentaram menor PG (P =0,001), em comparação com novilhas (10,42%). Na estação primavera/verão a P/TETF60 foi menor (P = 0,024). Maior PG tendeu a ocorrer na estação mais quente. O tratamento com gonadorelina no momento do TETF aumentou a prenhez por TE aos dias 30 e 60 e reduziu o PG. Vacas receptoras secas e lactantes apresentaram menor taxa de PG em comparação com novilhas. Além disso, a TE realizada nas estações mais quentes do ano resultou em menor taxa de prenhez aos 60 dias e maior PG.

Palavras-chave:
eficiência reprodutiva; gado de leite; reprodução

1. Introduction

Embryo transfer (ET) is an important biotechnology for animal production, constituting an important tool for the multiplication of animals of superior genetic merit(¹1 Batista EOS, Guerreiro BM, Freitas BG, Silva JCB, Vieira LM, Ferreira RM, Rezende RG, Basso AC, Lopes RNVR, Rennó FP. Plasma anti-Müllerian hormone as a predictive endocrine marker to select Bos taurus (Holstein) and Bos indicus (Nelore) calves for in vitro embryo production. Domestic Animal Endocrinology. 2016; 54, 1–9. https://doi. org/10.1016/j.domaniend.2015.08.001
https://doi. org/10.1016/j.domaniend.201... ). According to Viana(²2 Viana JHM. 2020 Statistics of embryo production and transfer in domestic farm animals. Embryo Technology Newsletter. 2022; 39, 24–38), in 2020 more than 1.1 million in vitro embryos were produced worldwide. This shows great potential and the importance of this technique in cattle breeding(²2 Viana JHM. 2020 Statistics of embryo production and transfer in domestic farm animals. Embryo Technology Newsletter. 2022; 39, 24–38). Despite the growth in the usage of ET in cattle, its results remain inconsistent. Recipients of in vitro embryo production (IVEP) have lower conception rates and greater gestational loss compared to cows receiving in vivo-produced embryos and artificial insemination(³3 Sartori R, Prata AB, Figueiredo ACS, Sanches BV, Pontes GCS, Viana JHM. Update and overview on assisted reproductive technologies (arts) in Brazil. Anim. Reprod. 2016; 13, 300–312. http://dx.doi.org/10.21451/1984-3143-AR873
http://dx.doi.org/10.21451/1984-3143-AR8... ). Therefore, studies should be developed to discover new strategies to increase the efficiency of in vitro embryo production and transfer programmes.

Among several factors that can influence the success of ET, the quality of the corpus luteum (CL) plays an important role in pregnancy maintenance(⁴4 Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reproduction in Domestic Animals. 2008; 43, 260–267. https://doi.org/10.1111/j.1439-0531.2008.01171.x
https://doi.org/10.1111/j.1439-0531.2008... ). Corpus luteum quality is correlated with serum progesterone (P4) concentration, which is responsible for conception and intrauterine embryo development(⁵5 Wiltbank MC, Souza AH, Carvahlo PA, Cunha AP, Giordano JO, Fricke PM. Physiological and practical effects of progesterone on reproduction in dairy cattle. Animal. 2014; 8(1), 70–81. https://doi.org/10.1017/ S1751731114000585
https://doi.org/10.1017/ S17517311140005... ). The increase in serum P4 concentration immediately after insemination has a function in maintaining pregnancy and promoting proper development of the conceptus(⁶6 Inskeep EK. Preovulatory, postovulatory, and post maternal recognition effects of concentrations of progesterone on embryonic survival in the cow. Journal of Animal Science. 2004; 82, 24–39. https://doi.org/10.2527/2004.8213_ supplE24x
https://doi.org/10.2527/2004.8213_ suppl... ,⁷7 Stronge AJH, Sreenan JM, Diskin MG, Mee JF, Kenny DA, Morris DG. Post-insemination milk progesterone concentration and embryo survival in dairy cows. Theriogenology. 2005; 64, 1212–1224. https://doi.org/10.1016/j. theriogenology.2005.02.007
https://doi.org/10.1016/j. theriogenolog... ,⁸8 Mcneil RE, Diskin MG, Sreenan JM, Morris DG. Associations between milk progesterone on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology. 2006; 65, 1435–1441. https://doi. org/10.1016/j.theriogenology.2005.08.015
https://doi. org/10.1016/j.theriogenolog... ). Indirectly, the presence of P4 in the uterine environment modifies endometrial gene expression(⁹9 Lonergan P, Forde N. Maternal-embryo interaction leading up to the initiation of implantation of pregnancy in cattle. Animal. 2014; 8, 64–69. https://doi.org/10.1017/S1751731114000470
https://doi.org/10.1017/S175173111400047... ), which in turn enables the development of the embryo and the production of interferon tau. The direct effect of P4 on embryo elongation has not been fully elucidated. However, the presence of P4 at correct levels, even before embryo deposition through inovulation, prepares the uterine environment, making it more receptive to the embryo and enhancing its development(¹⁰10 Geisert RD, Short EC, Zavy MC. Maternal recognition of pregnancy.1991; 282, 87–298.).

Postovulatory treatment with luteotropic hormones in the first week of the oestrous cycle, such as human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH), can improve pregnancy rates and reduce pregnancy losses(¹¹11 Marques MO, Nasser LF, Silva RCP, Bó GA, Sales JNS, Sá Filho MF, Reis EL, Binelli M, Baruselli PS. Follicular dynamics and pregnancy rates in Bos taurus x Bos indicus embryo transfer recipients treated to increase plasma progesterone concentrations. Anim. Reprod. 2012; 9, 111–119.,¹²12 Vasconcelos JLM, Sá Filho OG, Justolin PLT, Morelli P, Aragon FL, Veras MB, Soriano S. Effects of post breeding gonadotropin treatments on conception rates of lactating dairy cows subjected to timed artificial insemination or embryo transfer in a tropical environment. Journal of Dairy Science. 2011; 94, 223–234. https://doi.org/10.3168/ jds.2010-3462
https://doi.org/10.3168/ jds.2010-3462... ). These hormones promote the formation of an accessory CL and, consequently, an increase in serum P4 concentration. Another explanation for the increase in pregnancy rate has been described in cows treated with luteotropic hormones. The higher P4 concentration, caused by the formation of an accessory CL, leads to better modulation of follicular growth. Thus, the follicles growing under these conditions are usually smaller and, therefore, produce less oestrogen. At this time, high concentrations of oestrogen are not desirable since it can increase the possibility of luteolysis, which would interrupt the pregnancy(¹¹11 Marques MO, Nasser LF, Silva RCP, Bó GA, Sales JNS, Sá Filho MF, Reis EL, Binelli M, Baruselli PS. Follicular dynamics and pregnancy rates in Bos taurus x Bos indicus embryo transfer recipients treated to increase plasma progesterone concentrations. Anim. Reprod. 2012; 9, 111–119.). Due to the inconsistent results of fixed-time embryo transfer (FTET) and the controversial effectiveness of luteotropic hormones in increasing the pregnancy rate(¹³13 Loiola MVG, Pereira DFC, Vasconcelos LV, Lima MCC, Ferraz PA, Rodrigues A, Bittencourt RF, Jesus EO, Ribeiro Filho AL. Taxa de gestação de receptoras de embriões bovinos tratadas com um análogo do GnRH no momento da inovulação. Rev. Bras. Saúde Prod. Anim. 2014; 15, 782–789.), more research in this area is required.

The objectives of our study were to evaluate the effects of recipient category, season of the year and administration of a gonadorelin analogue (Fertagyl®) at the time of FTET on pregnancy per FTET at 30 (P/FTET 30) and 60 (P/FTET 60) days after oestrus and on pregnancy losses (PL).

2. Material and methods

The study was carried out on 11 commercial farms in Uberlândia, Minas Gerais state, Southeast Brazil, from July 2015 to July 2016. According to the Köppen classification, the climate is the Aw type, megathermal, with hot and rainy summers (from October to March) and cold and dry winters (from April to September)(¹⁴14 Rosa R. Abordagem preliminar das condições climáticas de Uberlândia-MG. Sociedade & Natureza. 1991; 3, 91–108.). Average temperature and relative humidity were 24.97 °C and 69.05% in the spring/summer seasons and 22.29 °C and 49.34% in the autumn/winter seasons, respectively(¹⁵15 IGUFU – Instituto de Geografia da Universidade Federal de Uberlândia. 2017. Localizado em: Av. João Naves de Ávila, 2121 – Bloco 1H – Sala 1H 18A, Campus Santa Mônica – Uberlândia – MG – CEP 38400-902.). This study was conducted according to the Ethical Principles in Animal Experimentation, approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal University of Uberlândia (UFU) under approval number 041/14.

2.1 Donors and embryos

The study used 144 oocyte donors and semen from 38 different sires. The donors did not undergo any hormonal protocol, and their oocytes were collected using the ovum pick-up method (OPU). The interval between OPU in a given donor was at least two months. All the embryos were produced in vitro. Embryos were classified by laboratory technicians according to their stage of development (from 1 to 9) and their morphological quality (from 1 to 4)(²2 Viana JHM. 2020 Statistics of embryo production and transfer in domestic farm animals. Embryo Technology Newsletter. 2022; 39, 24–38). Only embryos graded 1 for morphological quality and in developmental stages three (morula), four (compact morula), five (early blastocyst), six (blastocyst), seven (expanded blastocyst) or eight (hatched blastocyst) were used(¹⁶16 Bó GA, Mapletoft, RJ. Evaluation and classification of bovine embryos. Anim. Reprod. 2013; 10, 168–173.). Nellore (80), Gyr (530) and Girolando (701) embryos were used. The embryos were transferred fresh according to the order in which they were allocated in the embryo carrier.

2.2 Recipients and treatment

To be eligible for the FTET programme, crossbred recipients could not show any clinical disease. In addition, a body condition score (BCS) ranging between 3 and 3.75, on a scale from 1 to 5(¹⁷17 Klopčič M, Hamoen A, Bewley J. Body condition scoring of dairy cows. University of Ljubljana Biotechnical Faculty. Slovenia. 2011; 1–44.), was required. The selected recipients were randomly divided into two groups: the treated group (n = 624, treated with 0.2 mg of gonadorelin (Fertagyl) on the day of FTET; and the control group (n = 687, not treated).

The hormonal protocol used to synchronise the recipients’ ovulation was: D0: insertion of the intravaginal P4 (FertilCare®) device at a dose of 1.2 g and intramuscular application of 2.0 mg of oestradiol benzoate (FertilCare Sincronização®); D7: administration of 0.526 mg of sodium cloprostenol (Sincrosin®) i.m. (intramuscularly); D9: administration of 0.526 mg of sodium cloprostenol (Sincrosin®) i.m. + 1 mg of oestradiol cypionate (FertilCare Ovulação®) i.m. + removal of the intravaginal device; D18: recipients underwent a transrectal ultrasound examination and only those that had at least one CL were considered eligible for the FTET programme. The embryos were transferred to the ipsilateral uterine horn of the ovary that had the CL, and administration or omission of 0.2 mg of gonadorelin i.m. (Fertagyl®) was carried out. Embryos were designated to recipients randomly. Evaluations of health, BCS and ovaries, as well as all inovulations and pregnancy diagnoses, were performed by the same veterinarian.

2.3 Pregnancy diagnoses and data collection

For pregnancy diagnosis, a transrectal ultrasound examination was performed 23 ± 5 days after FTET. Around 30 ± 5 days later, animals diagnosed as pregnant were re-examined. Pregnancy per FTET at 30 days of gestation (P/FTET 30) was determined as the ratio between the number of pregnant animals in the first evaluation and the total number of FTET. Pregnancy per FTET at 60 days of gestation (P/FTET 60) was calculated as the ratio between the number of pregnant animals in the second evaluation and the total number of FTET. Finally, pregnancy loss (PL) was calculated by the ratio between the number of pregnancy losses between the first and second diagnoses and the number of pregnant animals in the first diagnosis. The result of these divisions was multiplied by 100 to obtain a percentage value. Data on the season of the year when the FTET was performed (spring/summer and autumn/winter) and recipient category (heifers, lactating cows and dry cows) were also collected.

2.4 Statistical analysis

The data with binomial distribution (P/FTET 30, P/FTET 60 and PL) were analysed by multivariate logistic regression, using the GLIMIX procedure of SAS version 9.2 (SAS/STAT, SAS Institute Inc., Cary, NC). In the first model, the effects of treatment, FTET season of the year, recipient category, as well as possible interaction effects on P/FTET 30, P/FTET 60 and PL were evaluated. When no effect of the possible interactions was detected, they were removed from the final model. Statistical significance was considered when P ≤ 0.05, and a statistical tendency when 0.05 < P ≤ 0.10.

3. Results

The treatment affected both P/FTET 30 (P = 0.03) and P/FTET 60 (P = 0.01). The treatment with gonadorelin on day 7 tended (P = 0.09) to reduce PL (7% and 4% for control and treated groups, respectively) (Table 1).

There was no effect of the recipient category on P/FTET 30 and P/FTET 60; however, heifers showed higher PL (P = 0.001 ) than dry cows and lactating cows (Table 2). The season of FTET affected pregnancy per FTET at 60 days (P = 0.024) and tended to affect (P = 0.095) PL. During the spring/summer season, PL tended to be greater compared to the autumn/ winter. Consequently, pregnancy per ET at 60 (P/ET 60) was higher in the autumn/winter seasons (Table 3).

4. Discussion

One of the aims of this study was to evaluate whether treatment with gonadorelin at the time of ET would improve pregnancy per FTET at 30 and 60 days or not. The results showed that the groups treated with gonadorelin at the time of FTET had increased pregnancy outcomes when compared to the control group (5.8% at 30 days and 6% at 60 days; Table 1). Similar results were found by Marques et al.(¹¹11 Marques MO, Nasser LF, Silva RCP, Bó GA, Sales JNS, Sá Filho MF, Reis EL, Binelli M, Baruselli PS. Follicular dynamics and pregnancy rates in Bos taurus x Bos indicus embryo transfer recipients treated to increase plasma progesterone concentrations. Anim. Reprod. 2012; 9, 111–119.), in which animals treated with GnRH had a higher P/ET compared to the control group. Vasconcelos et al.(¹²12 Vasconcelos JLM, Sá Filho OG, Justolin PLT, Morelli P, Aragon FL, Veras MB, Soriano S. Effects of post breeding gonadotropin treatments on conception rates of lactating dairy cows subjected to timed artificial insemination or embryo transfer in a tropical environment. Journal of Dairy Science. 2011; 94, 223–234. https://doi.org/10.3168/ jds.2010-3462
https://doi.org/10.3168/ jds.2010-3462... ) also found a higher P/ET in recipients treated with hCG and GnRH, compared to the control group. Together, these studies show that the administration of an ovulation inducer can be efective in improving pregnancy outcomes in ET programmes.

According to Guerra et al.(¹⁸18 Guerra AG, Sala RV, Sala LC, Baez GM, Motta JCL, Fosado M, Moreno JF, Wiltbank MC. Postovulatory treatment with GnRH on day 5 reduces pregnancy loss in recipients receiving an in vitro produced expanded blastocyst. Theriogenology. 2020; 14, 202–210. https://doi.org/10.1016/j.theriogenology.2019.05.010
https://doi.org/10.1016/j.theriogenology... ), treatment with gonadorelin, causing the dominant follicle of the first follicular wave to ovulate and form an accessory CL, can improve recipient P/ET. This treatment causes an increase in P4 concentration(¹⁹19 Cabrera EM, Lauber MR, Peralta EM, Bilby TR, Fricke PM. Human chorionic gonadotropin dose response for induction of ovulation 7 days after a synchronized ovulation in lactating Holstein cows. Journal of Dairy Science communications. 2021; 2(1), 35-40. https://doi.org/10.3168/jdsc.2020-0024
https://doi.org/10.3168/jdsc.2020-0024... ), which is of fundamental importance for the development of the conceptus and maintenance of pregnancy. Additionally, the high concentration of P4 provides a more favourable uterine environment for the elongation of the conceptus, which positively influences the production of interferon tau and the maternal recognition of pregnancy(²⁰20 Maillo V, Rizos D, Besenfelder U, Havlicek V, Kelly AK, Garrett M, Lonergan P. Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows. Journal of Dairy Science. 2012; 95, 3865–3876. https://doi.org/10.3168/jds.2011-5270
https://doi.org/10.3168/jds.2011-5270... ).

A second possible explanation for the results found is that ovulation of the dominant follicle in the first follicular wave leading to the formation of an accessory CL and the consequent increase in the P4 concentration inhibit the presence of a new large dominant follicle at the time of maternal pregnancy recognition, reducing the possibility of elevating circulating oestradiol and the development of the luteolytic trigger at this critical period of pregnancy(¹¹11 Marques MO, Nasser LF, Silva RCP, Bó GA, Sales JNS, Sá Filho MF, Reis EL, Binelli M, Baruselli PS. Follicular dynamics and pregnancy rates in Bos taurus x Bos indicus embryo transfer recipients treated to increase plasma progesterone concentrations. Anim. Reprod. 2012; 9, 111–119.). A study that characterized follicle and luteal dynamics and luteolysis in non-inseminated lactating Holstein cows that form accessory CL due to treatment with hCG after ovulation indicated that forming accessory CL with the use of hCG disrupted natural luteolysis and ovulation subsequent to the treatment. These are critical physiological events that regulate oestrous cycle length, but a possible delay in luteolysis could be beneficial to the pregnancy outcomes after ET(²¹21 Cunha TO, Statz, LR, Domingues RR, Andrade JPN, Wiltbank MC, Martins JPN. Accessory corpus luteum induced by human chorionic gonadotropin on day 7 or days 7 and 13 of the estrous cycle affected follicular and luteal dynamics and luteolysis in lactating Holstein cows. Journal of Dairy Science.2022; 105(3), 2631-2650. https://doi. org/10.3168/jds.2021-20619
https://doi. org/10.3168/jds.2021-20619... ).

In contrast, some studies have shown that the use of drugs that promote the formation of an accessory CL does not improve pregnancy in recipients(¹³13 Loiola MVG, Pereira DFC, Vasconcelos LV, Lima MCC, Ferraz PA, Rodrigues A, Bittencourt RF, Jesus EO, Ribeiro Filho AL. Taxa de gestação de receptoras de embriões bovinos tratadas com um análogo do GnRH no momento da inovulação. Rev. Bras. Saúde Prod. Anim. 2014; 15, 782–789.). Guerra et al.(¹⁸18 Guerra AG, Sala RV, Sala LC, Baez GM, Motta JCL, Fosado M, Moreno JF, Wiltbank MC. Postovulatory treatment with GnRH on day 5 reduces pregnancy loss in recipients receiving an in vitro produced expanded blastocyst. Theriogenology. 2020; 14, 202–210. https://doi.org/10.1016/j.theriogenology.2019.05.010
https://doi.org/10.1016/j.theriogenology... ) did not find any improvement in the conception rate of animals treated with GnRH. According to the authors, this may have occurred because both groups, treated and control, had high fertility and low PL. Therefore, GnRH treatment 5-7 days after ovulation may not be efficient in herds with good fertility results.

Loiola et al.(¹³13 Loiola MVG, Pereira DFC, Vasconcelos LV, Lima MCC, Ferraz PA, Rodrigues A, Bittencourt RF, Jesus EO, Ribeiro Filho AL. Taxa de gestação de receptoras de embriões bovinos tratadas com um análogo do GnRH no momento da inovulação. Rev. Bras. Saúde Prod. Anim. 2014; 15, 782–789.) reported a lower pregnancy rate for a group treated with GnRH. However, the protocol they used included administration of equine chorionic gonadotrophin (eCG). According to Baruselli et al.(²²22 Baruselli PS, Reis EL, Carvalho NAT, Carvalho JBP. eCG increase ovulation rate and plasmatic progesterone concentration in Nelore (Bos indicus) heifers treated with progesterone releasing device. Anais. Belo Horizonte, MG. 2004.), the protocol used for recipient synchronisation can influence the P/ET. Protocols that use eCG usually lead to a greater development of the dominant follicle and, consequently, to the formation of a larger CL. A larger CL has an increased capacity for P4 production, leading to higher pregnancy rates. The use of eCG can also increase the double ovulation rate when administered during the predominant phase of the follicular wave, increasing the P4 concentrations after ovulation. Since the use of eCG already increases the P4 concentrations, treatment with GnRH after ovulation may not influence P/ET rates in this case. This may explain the results found by Loiola et al. (¹³13 Loiola MVG, Pereira DFC, Vasconcelos LV, Lima MCC, Ferraz PA, Rodrigues A, Bittencourt RF, Jesus EO, Ribeiro Filho AL. Taxa de gestação de receptoras de embriões bovinos tratadas com um análogo do GnRH no momento da inovulação. Rev. Bras. Saúde Prod. Anim. 2014; 15, 782–789.), which diverge from those of the present study.

The treatment with gonadorelin tended to reduce the PL rate (P = 0.095; Table 1). Some studies that administrated different luteotropic drugs found similar results to the present study(²³23 Niles AM Fricke HP, Carvalho PD, Wiltbank MC, Hernandez LL, Fricke PM. Effect of treatment with human chorionic gonadotropin 7 days after artificial insemination or at the time of embryo transfer on reproductive outcomes in nulliparous Holstein heifers. Journal of Dairy Science. 2019; (102), 2593–2606. https://doi.org/10.3168/ jds.2018-15588
https://doi.org/10.3168/ jds.2018-15588... ). Niles et al.(²³23 Niles AM Fricke HP, Carvalho PD, Wiltbank MC, Hernandez LL, Fricke PM. Effect of treatment with human chorionic gonadotropin 7 days after artificial insemination or at the time of embryo transfer on reproductive outcomes in nulliparous Holstein heifers. Journal of Dairy Science. 2019; (102), 2593–2606. https://doi.org/10.3168/ jds.2018-15588
https://doi.org/10.3168/ jds.2018-15588... ) observed a reduction in PL between the first and second pregnancy diagnoses (days 32 to 67) in heifers treated with hCG on the day of TFET (seven days after ovulation). Guerra et al.(¹⁸18 Guerra AG, Sala RV, Sala LC, Baez GM, Motta JCL, Fosado M, Moreno JF, Wiltbank MC. Postovulatory treatment with GnRH on day 5 reduces pregnancy loss in recipients receiving an in vitro produced expanded blastocyst. Theriogenology. 2020; 14, 202–210. https://doi.org/10.1016/j.theriogenology.2019.05.010
https://doi.org/10.1016/j.theriogenology... ), studying the administration of GnRH on day 5 of the oestrous cycle of Holstein and crossbred heifers, reported an effect of embryo quality on the rate of PL. Animals that received embryos in development stage 7 (expanded blastocyst) had a lower percentage of pregnancy losses than the other recipients. In addition, the effect of the presence of an accessory CL in these animals potentiated the reduction of PL. This reduction in PL reached up to 50%. According to the authors, the embryo stage and the presence of an accessory CL (which is responsible for higher P4 concentrations) are factors that possibly contributed to the decrease in PL.

An effect of the recipient categories (heifers, dry cows and lactating cows) on pregnancy per ET at 30 days and 60 days was not detected; however, heifers showed greater PL when compared to the other groups (P = 0.001; Table 2). These results were contrary to what was expected since most data in the literature show that heifers tend to have better pregnancy outcomes and fewer pregnancy losses(²⁴24 Hasler JF. Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America, and personal reminisces. Theriogenology. 2014; 81, 152–169. https://doi. org/10.1016/j.theriogenology.2013.09.010
https://doi. org/10.1016/j.theriogenolog... ). However, the PL rate for heifers in this study met the values found in other studies(²⁵25 Scanavez AL, Campos CC, Santos RM. Taxa de prenhez e de perda de gestação em receptoras de embriões bovinos produzidos in vitro. Arq. Bras. Med. Vet. Zootec. 2013; 65, 722–728. https://doi.org/10.1590/S0102-09352013000300017
https://doi.org/10.1590/S0102-0935201300... ). Thus, this diference may be due to the low PL rate found for the other two categories of recipients (dry and lactating cows). Normally, reduced P/ET and PL rates are observed for high-producing lactating cows(²⁶26 Vasconcelos JLM, Demétrio DGB, Santos RM, Chiari JR, Rodrigues CA, Filho OGS. Factors potentially affecting fertility of lactating dairy cow recipients. Theriogenology. 2006; 65, 192–200. https://doi.org/10.1016/j. theriogenology.2005.09.030
https://doi.org/10.1016/j. theriogenolog... ). Since the recipient cows used in the present study were either low-producing or dry cows the negative effect of milk production may not have impacted the PL rates, thus explaining the low values found.

A study that used hCG as an ovulation inducer revealed a higher concentration of progesterone on day 12 after AI, and, regardless of parity, pregnancy was improved only in the primiparous cows, compared to the multiparous cow(²⁷27 Zolini AM, Ortiz WG, Estrada-Cortes E, Ortega MS, Dikmen SERDAL, Sosa F, Giordano JO, Hansen PJ. Interactions of human chorionic gonadotropin with genotype and parity on fertility responses of lactating dairy cows. Journal of Dairy Science. 2019;102(1), 846-856. https://doi.org/10.3168/jds.2018-15358
https://doi.org/10.3168/jds.2018-15358... ). In contrast with the present results, Silva et al. (2023)(²⁸28 Silva LO, Motta JC, Oliva AL, Madureira G, Alves RL, Folchini NP, Silva MA, Silva TJB, Consentini CEC, Wiltbank MC, Sartori R. Influence of GnRH analog and dose on LH release and ovulatory response in Bos indicus heifers and cows on day seven of the estrous cycle. Theriogenology. 2023. https://doi.org/10.1016/j.theriogenology.2023.10.015
https://doi.org/10.1016/j.theriogenology... ) observed that a single dose of GnRH administration was able to induce ovulation in Bos indicus heifers, but not in cows. According to the authors, a possible reason for this finding is that ovulation in heifers may require a lower LH peak compared to cows, leading to a possible parity effect at the follicular level impacting the ovulatory response.

Season of the year was found to affect (P = 0.024) pregnancy per ET at 60 days. Recipients that underwent FTET in the autumn/winter showed better reproductive performance compared to animals in which FTET was performed in the spring/summer (Table 3). Demétrio et al.(²⁹29 Demétrio DG, Santos RM, Demetrio CG, Vasconcelos JL. Factors affecting conception rates following artificial insemination or embryo transfer in lactating Holstein cows. Journal of Dairy Science. 2007; 90, 5073–5082. https:// doi.org/10.3168/jds.2007-0223
https:// doi.org/10.3168/jds.2007-0223... ) reported that an increase in the body temperature of animals on the seventh day after ovulation has a negative effect on pregnancy at 28 days in animals subjected to ET. Similarly, Ferraz et al.(³⁰30 Ferraz PA, Burnley C, Karanja J, Vieira-Neto A, Santos JEP, Chebel RC, Galvão KN. Factors affecting the success of a large embryo transfer program in Holstein cattle in a commercial herd in the southeast region of the United States. Theriogenology. 2016; 86, 1834–1841. https://doi.org/10.1016/j.theriogenology.2016.05.032
https://doi.org/10.1016/j.theriogenology... ) evaluated the relationship between the temperature-humidity index (THI) and the conception rate and observed that the conception rate for cows and heifers decreased with the increase in THI. They also found that cows are more sensitive to heat stress indicated by the THI compared with heifers. According to Sartori et al. (³¹31 Sartori R, Sartor-Bergfelt R, Mertens SA, Guenther JN, Parrish JJ, Wiltbank MC. Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. Journal of Dairy Science. 2002; 85, 2803–2812. https://doi.org/10.3168/jds.S0022-0302(02)74367-1
https://doi.org/10.3168/jds.S0022-0302(0... ), compared to heifers, cows have greater difficulty regulateing their body temperature in response to warm weather. This greater susceptibility to heat stress contributes to lower conception rates during the warm seasons.

In the present study, the season of the year in which FTET was performed tended to affect PL (P = 0.095). During the warmer seasons (spring/summer), PL was greater than in the colder seasons (autumn/winter) (Table 3). Garcia-Ispierto et al. (³²32 Garcia-Ispierto I, López-Gatius F, Santolaria, P, Yániz, J. L, Nogareda C, López-Béjar M, De Rensis F. Relationship between heat stress during the peri-implantation period and early fetal loss in dairy cattle. Theriogenology, Estados Unidos. 2006; 65(4), 799–807. https://doi.org/10.1016/j.theriogenology.2005.06.011
https://doi.org/10.1016/j.theriogenology... ) also evaluated the influence of heat stress on PL. They also observed a greater PL rate during the warmer months when compared to the colder periods. According to the authors, a high THI during the fourth week of gestation can negatively impact pregnancy rate and contribute to PL due to the embryo’s sensitivity to heat stress during the implantation period. Nanas et al. (2021)(³³33 Nanas I, Chouzouris TM, Dovolou E, Dadouli K, Stamperna K, Kateri I, Barbagianni M, Amiridis S.G. Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows. Journal of thermal biology. 2021; 98, 102951. https://doi.org/10.1016/j.jtherbio.2021.102951
https://doi.org/10.1016/j.jtherbio.2021.... ) analysed artificially inseminated cows and found a higher pregnancy rate in winter compared to summer. They also found greater PL in the summer. According to them, these results may be mainly due to luteal insufficiency. Based on this, we can infer that the treatment with GnRH after ET was not sufficient to prevent PL during the summer.

5. Conclusion

The treatment with gonadorelin at the time of bovine embryo transfer increased pregnancy per embryo transfer at days 30 and 60 and reduced pregnancy losses. Additionally, dry and lactating recipient cows showed a lower PL rate compared to heifers. Furthermore, embryo transfer performed in the warmer seasons of the year (spring/summer) resulted in a lower pregnancy rate at day 60 and a higher pregnancy loss rate.

Acknowledgements

We acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Research Productivity Grant – R. M. Santos. This study was financed in part by the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

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