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INTRODUCTION
Currently ET in cattle is a well established industry in Mexico and around the world, with 573,970 embryos transferred per year (Hasler 2014). The objective of ET focuses on genetic improvement of bovine cattle, producing meat, milk and dual purpose (Galli et al. 2003, Mapletoft 2013 and Bó et al. 2016). In recent years, genetic improvement in cattle has made great progress thanks to reproductive biotechnologies, artificial insemination (AI) and ET (König et al. 2007, Carvalho et al. 2008 and Bó et al. 2016). To implement AI and ET techniques, estrogen/estral synchronization protocols are used with synthetic hormones such as progestins and prostaglandin F2α (PGF2α) (Armstrong 1993 and Bó et al. 2016). In turn, these hormones can be combined with gonadotropin-releasing hormone (GnRH), estradiol, follicle- stimulating hormone (FSH) or equine chorionic gonadotropin (eCG). This combination results in shorter estrus and a suitable ovulation (Galli et al. 2003 and Núñez-Olivera et al. 2018). In this way, there is the possibility of having suitable cows for ET and AI at fixed time (with or without estrus detection) in a shorter period of time (Stevenson et al. 2000 and Núñez-Olivera et al. 2018).
The percentage of gestation in cows subjected to estrus synchronization and ET ranges between 40 and 70 % of efficiency (Hasler 2001). These differences could be explained by genetic, maternal and environmental factors. For example, Bényei et al. (2006), when performing ET in cows from a tropical region, recorded 43 % of gestation during the rainy season (September-April) and 38 % during dry season (May-August). On the other hand, Nasser et al. (2011) transferred embryos in Nelore cows and did not find differences in the gestation percentage among multiparous (48.1 %) and nulliparous (45.8 %) cows. In contrast, Hasler et al. (2001) when using fresh ET technique found a higher percentage of pregnancy in multiparous cows than in the nulliparous (79.9 % and 69.9 %). In this sense, success of ET programs used in cows producing meat, milk and dual purpose depends on different factors like if transferred embryos are "fresh" or cryopreserved, age of embryos, degree of estrus synchrony between the donor cow and the recipient, season and the experience of the technician (Hasler 2001, Peixoto et al. 2007, Bó et al. 2016 and Ferraz et al. 2016). Likewise, parity of recipient cows is essential to increase the probability of success of ET programs applied to cattle producing meat, milk and dual purpose (Ferraz et al. 2016). Therefore, the objective of this study was to evaluate the effect of parity on the reproductive efficiency of Suizo Americano cows subjected to a synchronization protocol and fresh ET.
MATERIALS AND METHODS
General conditions. The experimental procedures were carried out according to the official Mexican norms (NOM-062-ZOO-1999), with technical specifications for the production, use and care of laboratory animals (SAGARPA 2001). The standards described in the manual of the International Embryo Transfer Society 2007 were also taken into consideration.
The present study was carried out during the winter in the ranch "Las Tortolitas", Ometepec municipality, Guerrero, Mexico. This region is located in the tropics (16°43'26"N, 99°07'24"W), with temperatures higher than 34 °C during the summer (May-August) and minimum temperatures of 19 °C during the winter (December-April), and an average annual rainfall of 1.100 mm (García 1973).
Pre-experimental management of cows. Before starting the experiment, 99 cows of Suizo Americano breed (60 multiparous and 39 nulliparous) of a commercial herd were dewormed (ivermectin, Sanfer, Mexico D.F.) and vitamins (A, D and E, vigantol, Bayer, Mexico D.F.) were provided. Additionally, rectal ultrasonography was performed using portable ultrasound equipment equipped with a 7.5 MHz rectal transducer (Aloka SSD-500) to detect pregnancy and observe the ovarian structures. During the management, it was verified that cows were clinically healthy and without reproductive problems. Then, the experimental units were selected and the hormonal management and ET were performed.
Treatments and food. In the study, 26 recipient Suizo Americano cows, with an average weight of 522 ± 72 kg and body condition of 3.8 ± 0.26 units (scale 1-5 units, Edmonson et al. 1989) were used, which were divided, under a completely random design, into two experimental groups (n = 13) according to their parity (multiparous and nulliparous). Cows were grazing in paddocks with Tanzania grass (Panicum maximum) and star grass (Cynodon nlemfuensis) during the day (0500-1700 h) and during the evening (1700-0500 h) they were kept in open pens where they were provided with maize silage supplementation (0.5 kg per cow, table 1), blocks of mineral salts, and freely available water.
Reproductive management of cows. Superovulation protocol and AI of donor cows. Prior to the superovulation program, donor cows underwent the follicular wave synchronization to coincide the appearance of a new wave with the start of superovulation protocol. This consisted on application of 5 mg of estradiol benzoate (EB; Virbac®) for cows and 2 mg (Syntex®) for heifers, plus 100 mg progesterone (P4, Pfizer®) and the insertion of a controlled internal drug release (CIDR) device, containing 1.9 g of progesterone (P4, Zoetis®) on day zero. At the fifth day, superovulation started with the application of follicle stimulating hormone (FSH, Folltropin-V®), fractioned and decreasingly for four days in two daily applications with 12-hour intervals (2 applications of 60, 40, 20 and 20 mg). On the eighth day of treatment, the CIDR was withdrawn, and, on the ninth day of the beginning of superovulation, estrus were detected and each cow and/or heifer (12, 24 and 36 h) was inseminated with semen of Suizo Americano bulls.
Table 1 Chemical composition of aize silage (Zea mays) offered to recipient cows
Calculations performed under dry basis Total digestible nutrients (TDN=99.39- 0.7461NDF; Cappelle et al. 2001), energy (NRC, 1985): digestible (DE=TDN x 0.44), metabolizable (ME=0.82 x DE), net of maintanence (ENm=1.37 x ME - 0.14ME2 +0.01ME3 - 1.12) and net of gain (EN=1.42 x ME - 0.17ME2 + 0.012ME3 - 1.65).
Collection of embryos in donor cows. Each donor cow was placed in an immobilizing trap for the embryo collection. Once the cow was immobilized, the perianal area was washed and disinfected, 5-8 mL of 2% lidocaine hydrochloride was applied epidurally (Pisacaine® 2% vet, 20 mg lidocaine hydrochloride in 1 mL). When necessary, 2% xylazine was applied as a tranquilizer. Subsequently, a two-way Foley catheter was vaginally inserted, fixing it in the body of the uterus to perform infusions and uterine collections. An amount of 1.5 L of phosphate buffered solution, supplemented with glucose, pyruvate, antibiotics and 0.3% of polyvinyl alcohol (PBS, Dulbecco and Vogt 1954) was used. The filter contents were emptied in a 100 x 100 mm square Petri dish with a grid pattern for the subsequent search of embryos in the stereoscopic microscope with a magnification of 20 to 30X. The found embryos were placed in a 35 mm Petri dish with maintenance solution (PBS with bovine serum albumin). They were classified according to their morphology, stage of development on a scale of 1 (stage of a cell) to 9 (stage of hatched blastocyst) and according to their quality as 1 (excellent), 2 (good), 3 (regular) and 4 (degenerated) (International Embryo Transfer Society 2007).
Synchronization protocol and ET for recipient cows. To synchronize the estrus of recipient cows, a CIDR (Zoetis®) was intravaginally applied on day zero. On the fifth day, 2 mg of EB was administered intramuscularly (i.m.) to multiparous cows, 2.5 mg to nulliparous and 2.5 mg of luteinizing hormone (LH, Pluset®) to all cows. On the seventh day, all the cows received an intramuscular injection with 2 mg of FSH, 5 mg of LH, 0.5 mg of estradiol cypionate (EC, Zoetis®) and CIDR was withdrawn. On the eighth and ninth days, estrus was detected and ET was performed on the 15th day. Estrus detection was carried out from 6:00 to 6:30 h and from 18:00 to 18:30 h during two consecutive days, considering a female in estrus when she allowed two mounts during the observation period (Orihuela et al. 1989). Estrus cows were prepared for ET. Each female was immobilized and the perianal area was washed and disinfected, applying 5-8 mL of 2 % lidocaine hydrochloride epidurally. The ET technique was transcervical using a French applicator (Embryo transfer gun, Agtech 21 "Sheath, Radiated). The embryo was deposited in the distal third of the ipsilateral uterine horn where ovulation occurred. Gestation diagnosis of cows was performed with ultrasonography at 50 d post ET.
Statistical analysis. All the information was analyzed with the statistical program SYSTAT 13® (SYSTAT 13), under a completely at random design, where the cow was the experimental unit. In order to check the normal distribution of data from different variables, Kolmogorov-Smirnov, Lilliefors, normality test was used, and showed that most of the continuous data had a normal distribution. Therefore, parametric statistical tests were used. The reproductive variables gestation percentage, gestation by uterine horn (right vs. left), day of estrus, time of estrus and stage of the embryo (blastocyst and expanded blastocyst) were analyzed with the χ2 test. To compare means among groups of cows, the t-student test was used. Probabilities were considered as significant at P <0.05. All the results of the study were expressed as a percentage.
RESULTS AND DISCUSSION
The percentage of recipient cows with estrus was higher on the first day in multiparous cows than in nulliparous cows (P <0.05). However, the percentage of pregnant diagnosed cows was similar among groups (P> 0.24). In addition, the time of estrus in the morning and afternoon (table 2), and the stage of the blastocyst vs. expanded blastocyst embryo did not vary due to parity of cows (P> 0.05, figure 1). There was also no effect on the percentage of pregnancy between the right and left uterine horns, independently of the experimental group (P> 0.05, figure 1). The results of the present study show that multiparous cows had a greater response of estrus than nulliparous cows. In contrast, the percentage of pregnant cows, the time of estrus and the stage of the embryo were similar between multiparous and nulliparous cows.
Table 2 Reproductive performance of recipient cows that underwent fresh embryo transference
a,b Indicate significant differences within the same column (P<0.05)
x,y Indicate significant difference within the same line (P<0.05)
Fig. 1 Percentage of cows (multiparous▲ and nulliparous ●) that received embryos in stage of blastocyst or expanded blastocyst in the right or left horn. Results were not statistically significant among groups. In the superior part of shapes (▲, ●), P value is presented
Similar results were recorded by Chebel et al. (2008) (83 % of gestation), but different from that found by Ferraz et al. (2016), who reported in their study a higher percentage of pregnancy in nulliparous cows (42 %) than in multiparous cows (31.6 %). This difference in gestation percentage due to the effect of parity, probably due to the good functionality of the corpus luteum (CL) of the recipient cows (Siqueira et al. 2009). Although in this study progesterone levels in blood (P4) were not measured, results allow to infer that CL had an adequate secretion of this hormone. In addition, body condition of females demonstrates an adequate nutrition. Therefore, it is inferred that results were not affected by the nutritional state of these cows, regardless of parity.
Estrus response was superior in multiparous cows regarding nulliparous cows (92 % vs. 54 %) in the first 24 h after removing the CIDR. These results are similar to those found by Orihuela et al. (1989), who reported a 95 % estrus response in cows synchronized with a synthetic progestogen. Regarding estrus time, the results of this research are similar to those found by Orihuela et al. (1989), where more than 90 % of cows showed signs of estrus in the first 58 h after removing the CIDR. In contrast, Martínez-González et al. (2007) only recorded a 76 % of estrus response. This difference between the results of this study and those of these authors is probably because multiparous cows of the present study already had previous sexual experience, to the climatic conditions of winter, and, in general, to the good reproductive management applied to recipient cows in this study.
Gestation percentage due to parity in cows to that an embryo was transferred in the blastocyst stage did not differ with respect to those of the expanded blastocyst. Contrary to these results, Ferraz et al. (2016) found a lower gestation percentage in nulliparous cows (36.5 %) than in multiparous cows (38.1%). Consistent with the data recorded in this study, Ling et al. (1995) and Pérez-Durand et al. (2017) obtained a higher gestation percentage in cows when an early embryo morula and blastocyst stage (day 6 or 7 after artificial insemination) was transferred than in those cows that an older embryo was transferred. In this sense, the state of development and quality of embryos have an important function in the success of ET programs. In this regard, Görlach (1999) and Ferraz et al. (2016) mentioned that the best moment for collecting embryos after artificial insemination is day 7, in addition to being the appropriate time for practicing the fresh ET, the ideal stage of the embryo is expanded blastocyst.
Gestation percentage between uterine horns (right and left) was not affected by the parity of cows. Tervit et al. (1977) agreed with these results because they did not find differences between both uterine horns in their study. These authors did not evaluate the parity effect because the cows used were nulliparous and were not affected in the pregnancy, although the CL was found in the ovary opposite to where the pregnant horn was. This is interesting, since Fernández-Baca et al. (1973) pointed out that, in llamas, the highest gestation percentage occurs in the left horn, contrary to that of bovines, which occurs in the right horn (López-Gatius 1997). Although Fernández-Baca et al. (1979) mentioned that embryonic migration between uterine horns (right and left), it occurs because if the gestation occurs in the left horn, it may cause luteolysis of CL of the right ovary and end with gestation. Physiologically, this type of studies are interesting because of the participation of P4 in the maintenance of pregnancy, especially during the first days, which is the critical stage of embryonic implantation and it is the moment when it is decided whether gestation proceeds or not.
Finally, this research was conducted in a subhumid tropical region of Guerrero state during the winter, period in which the lowest environmental temperatures of the year (19 °C) are present. Therefore, it is suggested that cows subjected to ET program were not affected by heat stress. This shows some interesting research perspectives, such as comparing the variables studied at different times of the year (summer vs. winter), as well as evaluating the effect of other factors that may alter the reproductive response of cows such as: temperature-humidity index (THI) and caloric stress. These factors can negatively affect the reproductive response of multiparous and nulliparous cows (Putney et al. 1989 and Ferraz et al. 2016).
CONCLUSIONS
Results of this study allows to conclude that the parity does not affect the reproductive response of recipient Suizo Americano cows subjected to a ET program in tropical areas. Multiparous cows had a similar gestation percentage to nulliparous ones, but responded in greater proportion to estrus in the first 24 h after withdrawing the intravaginal device. Gestation percentage per uterine horn, time of estrus andstage of the embryo were not affected by the parity of cows (multiparous and nulliparous).
