Use of heterospermic semen in fixed-time artificial insemination of nelore cows

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1 REDVET - Revista electrónica de Veterinaria - ISSN Use of heterospermic semen in fixed-time artificial insemination of nelore cows ALMEIDA, Ricardo Garcia de 1, FARIA, Fábio José Carvalho 1, FERNANDES, Carlos Antônio de Carvalho 2, SANTOS, Marcelo Diniz dos 3, COSTA, Deiler Sampaio 1* 1 Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, UFMS, Campo Grande, MS. Brazil; 2 Universidade de Alfenas, UNIFENAS, Alfenas, MG. Brazil; 3 Universidade de Cuiabá, UNIC, Cuiabá, MT. Brazil; Corresponding author: deiler.costa@outlook.com ABSTRACT The objective of this study was to compare the effects of using conventional and heterospermic semen on the pregnancy rate of Nelore cows participating in a fixed-time artificial insemination (FTAI) program. The study used 506 Nelore cows, originating from two farms (farms 1 and 2) located in the State of Mato Grosso do Sul, Brazil. For the realization of FTAI, conventional semen (bulls A, B, and C) and heterospermic semen (HT; mixed semen of bulls A + B + C), from bulls of the Nelore breed, were used. The semen was evaluated for sperm morphology and tested for thermal resistance. The pregnancy rates obtained in farms 1 (40.52%) and 2 (65.33%) differed significantly (p<0.05). However, there was no difference between the pregnancy rates obtained from the semen of bulls A (54.05%), B (47.8%), C (54.2%), and HT (55.65%). Accordingly, it was concluded that the use of heterospermic semen did not increase the pregnancy rate of females inseminated in a fixed-time manner. Key words: sperm; nelore; pregnancy rate INTRODUCTION Fixed-time artificial insemination (FTAI) has contributed to an increase in the proportion of Brazilian cows that are artificially inseminated. Its use has allowed improved economy of labor, greater standardization of the lots of calves, concentration of the management of vaccination and weaning, shortening of the breeding season, concentration of the calving period, and increased profitability, with heavier weaned calves, due to the use of proven bulls (Madureira; Pimentel, 2005; Vasconcelos et al., 2006). Moreover, in 1

2 South America, especially in Brazil and Argentina, the commercial availability of diverse steroid and protein hormones has allowed the development of various hormonal protocols aimed at achieving better conception indices (Barros; Ereno, 2004). Several factors influence the results of an FTAI program, such as the body condition score (BCS) of the females (Meneghetti; Vasconcelos, 2008), animal category used (Azeredo et al., 2007), effect of the inseminator (Russi et al., 2010), and quality of the semen used (Arruda et al., 2005); to neglect any of these items can severely compromise the results in terms of the pregnancy rate. Evaluation of semen through additional tests has been the object of several surveys in an attempt to estimate the fertility of a bull (Correa et al., 1997; Mocé and Graham, 2008). However, despite these efforts, there still remains a great variation between bulls and semen samples from the same bull in the final conception rate of an FTAI program (Severo, 2009). In this sense, in an attempt to circumvent problems of low conception rates due to the quality of the semen of a particular bull, a number of alternatives have been proposed. Beatty et al. (1969) suggested that the fertility of the spermatozoa of an individual could be improved by its inclusion into a seminal mixture, and that the use of heterospermic semen could increase reproductive rates in commercial herds. Conversely, Azmal et al. (2004) report that no significant differences were found when comparing homospermic and heterospermic semen on the day of collection. However, when the samples were kept refrigerated at 4 C for three days, the seminal mixture showed higher values on the second and third post-collection days when compared to conventional semen. Nonetheless, to our knowledge, there is no previous study in the literature using heterospermic semen with the objective of attempting to increase the pregnancy rates in an FTAI program. Accordingly, the objective of this study was to compare the effects of using conventional and heterospermic semen on the pregnancy rate of Nelore cows participating in an FTAI program. MATERIAL AND METHODS The FTAI program was run on two farms located in the State of Mato Grosso do Sul, Brazil, during the breeding season (October-December). Farm 1 (n = 312) was located in the Pantanal plains (Pantanal do Rio Negro) and farm 2 (n = 354) was located in the highland region. All females underwent gynecological examination with the aid of an ultrasound device prior to the insemination. Therefore, the FTAI program comprised only multiparous cows in good reproductive conditions. All animals were in diestrus at the time of synchronization, and had between 30 and 65 days postpartum. No animal had received any insemination/natural service before FTAI. 2

3 The BCS of the cows was evaluated by the same technician in both farms, and only animals with a BCS between 2.0 and 4.0, on a scale from 1.0 to 5.0, were used (Wildman et al., 1982). All females were raised in an extensive system and were in good nutritional condition. In both farms the age of cows ranged from 4 to 8 years, and in farm 1 the mean body weight was 407 ± and in farm 2, 432 ± 36.72kg. For the realization of the FTAI, commercial conventional frozen semen (bulls A, B, and C) and commercial heterospermic frozen semen (mixed semen from bulls A + B + C) from bulls of the Nelore breed, were used. All straws from each bull used in this study were from same batch. Semen samples were subjected to a heat resistance test (HRT) or 3 hours at 37 C (Borges et al., 2011). Sperm morphology was determined by counting 200 cells, and using the classification system proposed by Blom (1973). Only the samples considered approved were used, based on standards established by the Brazilian College of Animal Reproduction (CBRA, 2013); the results are presented in Table 1. Table 1. Evaluation of homospermic and heterospermic semen samples used in the FTAI programs 3 Bull Breed Mot(%)/Vig(1- Sptz HRT % normal Conclusion 5) 1 (n) 2 A Nelore 40/4 14 x /3 87 Able B Nelore 35/4 14 x /3 83 Able C Nelore 40/4 14 x /4 91 Able HT 4 Nelore 35/4 14 x /3 90 Able 1 Post-thaw motility (Mot) and vigor (Vig); 2 number of viable spermatozoa per dose; 3 motility (%)/sperm vigor (1-5) after a thermal resistance test for three hours at 37 C; 4 heterospermic semen (Bull A + B + C). Sptz = spermatozoa; HRT = heat resistence test. Hormonal applications were performed by the same team on both farms and the protocol used for all animals was as follows: Day 0: intramuscular application (IM) of 2 mg of estradiol benzoate (Estrogin, Farmavet, Brazil) and introduction of an intravaginal progesterone implant (CIDR, Zoetis, Brazil); Day 7: IM application of 12.5 mg of dinoprost (prostaglandin F2alpha) (Lutalyse, Zoetis, Brazil); Day 9: withdrawal of the progesterone implant, IM application of 300 IU of equine chorionic gonadotropin (Novormon, MSD Animal Health, Brazil) and IM application of 0.6 mg of estradiol cypionate (ECP Zoetis, Brazil); and Day 11: artificial insemination without estrus observation. The inseminations were performed by two different inseminators each one inseminating a maximum of 20 animals, until being replaced by the other. 3

4 The same two inseminators were used in both farms. The used semen, conventional or heterospermic, was randomized for each cow, conforming to the animal's entry order in the containment frame. The diagnosis of pregnancy was performed via rectal palpation, with the assistance of ultrasonographic equipment (Mindray, model DP-2200Vet with a 7.5-MHz linear rectal transducer), 30 days after insemination. The experimental design was randomized and the results were subjected to a frequency dispersion test (Chi-squared), using the Bioestat package, and the adopted significance level was 5%. RESULTS AND DISCUSSION The pregnancy rates obtained in farms 1 (40.52%) and 2 (65.33%) differed significantly (p<0.05). This site effect was probably due to the management and nutritional conditions on each farm. Farm 1, which showed a lower pregnancy rate, was located in the Rio Negro Pantanal lowlands region and had a poorer quality of pastures at the time of the experiment, which probably influenced the observed rate. In the Pantanal lowlands region, the productivity of beef cattle raised extensively in native pastures is low. One of the main factors is due to the seasonality of pasture production, aggravated by the floods that depending on the duration and intensity, can leave submerged most of the areas for grazing, and the large number of invasive species (Pott, 1997). On the other hand, in the region of the highlands of Mato Grosso do Sul, where the farm 2, was located the pastures and the soil were of better quality and there are no floods. Usually, these local characteristics favor the greater precocity and genetic quality of the herds of the highlands region. Some experiments have shown that factors intrinsic to humans, such as ability, muscle strength, and human-animal interactions, may affect the results of the FTAI, and that, moreover, the competency of the staff, quality of life and interpersonal relationships are considered important factors influencing the performance of the inseminators (Russi et al., 2009; Russi et al., 2010). However, contrarily to what was reported in these previous studies, no effect of the inseminator on the pregnancy rate was observed (p>0.05) in the present study, and both presented similar results within the same farm (Table 2). This observation was likely due to the fact that both inseminators were experienced and had undergone previous training in the experiment to standardize all semen thawing procedures, preparation of the applicator, and insemination. 4

5 Table 2. Ratios of pregnant cows (mean ± standard deviation) by body condition scores (BCS) and inseminator in the FTAI program according to the farm studied Farm BCS Inseminator Pregnancy rate Total pregnancy rate ± 0.51 a ± 0.32 a % (46/116) a 41.38% (48/116) a 67.88% (93/137) b 62.77% (86/137) b Mean values followed by different letters in the same column differ significantly (p<0.05) % (94/232) a 65.33% (179/274) b In spite of the difference in the pregnancy rate between the two farms, there was no significant effect from the semen used (p>0.05). Other confounding factors may have resulted in this effect not being observed. The pregnancy rates obtained with conventional and heterospermic semen are summarized in Table 3. Table 3. Pregnancy rates in Nelore cows inseminated in a fixed time, with heterospermic (HT) or conventional semen, in the different farms Farm HT* Bull A Bull B Bull C (27/58) a 41.4 (24/58) a 36.2 (21/58) a 37.9 (22/58) a 64.7 (44/68) b 66.7 (46/69) b 59.4 (41/69) b 70.5 (48/68) b Mean values followed by the same letter on each line did not differ significantly (p>0.05 by the Chi-Square test). * HT = heterospermic semen of bulls (A + B + C). The pregnancy rate obtained in females inseminated with heterospermic semen did not differ from the rate observed in those inseminated with conventional semen (p>0.05). This result is in contrast with the report by Nelson et al. (1975), in which higher pregnancy rates in the groups using a seminal mix of three bulls were observed compared to the groups receiving a seminal mixture of two bulls or conventional semen. Moreover, Kushner (1954) in his review found several advantages of using heterospermic semen, including an increase in conception rates when compared to conventional semen. However, in an experiment in which the cows were inseminated with a fresh semen mixture from four bulls, there were no observed differences in the proportion of descendants of each bull (Stewart et al., 1974), whereas, 5

6 when this same mixture was frozen and the insemination was performed later, there was a significant difference (p<0.01) in respect to the descendants of each bull, indicating that some animals present a decrease in fertility when their semen is frozen. This difference between frozen heterospermic and conventional semen is probably due to the fact that the bull spermatozoa that are more resistant to freezing, when used in a seminal mix, can compensate for the spermatozoa that are less resistant to cryopreservation (Azmal et al., 2004). Other authors working with conventional artificial insemination found no difference in the quality of conventional semen when compared to heterospermic semen (Dott; Walton, 1958; Lamb et al., 2001; Gottschal et al., 2009). However, to our knowledge, there is no previous article relating to the use heterospermic semen in FTAI. Although there was no information on the individual fertility of each bull, there was no significant difference (p>0.05) in the pregnancy rates among the individual bulls (A, B, and C) in this study. This fact may have influenced the lack of difference in the pregnancy rate of animals inseminated with HT semen (A + B + C), since the fertility of heterospermic semen is directly related to the rate of conception of each homospermic semen (Beatty et al., 1969; Diziuk, 1995). In addition to these results, there were also no significant differences detected in the pregnancy rate in relation to the BCS (p> 0.05), as shown in Table 2. The BCS positively correlates with the proportion of body fat and with the luteinizing hormone level (Schillo et al., 1992), which has been demonstrated to improve the ovulation rate and, subsequently, the pregnancy rate, in FTAI programs (Baruselli et al., 2004). Several authors have highlighted the importance of BCS in animal reproduction and their effects on the pregnancy rate (Osoro; Wright, 1992; Vieira et al., 2005). In their retrospective analysis, Sá Filho et al. (2009) found a positive effect between BCS and the pregnancy rate in FTAI programs. However, despite the animals in farm 1 having a BCS numerically lower than that observed in farm 2, these values did not significantly differ in the present study (p>0.05). This result may have been due to the use of equine chorionic gonadotropin in the synchronization protocol, which increases the frequency and amplitude of luteinizing hormone pulses, thereby contributing to an increase in the pregnancy rate of cows with low BCS or in anestrous (Baruselli et al., 2004). The use of heterospermic semen, in our opinion, is a recommended technique to evaluate the fertility of a bull, as reported by Dziuk (1995). However, some researchers recommend its use when there are two or three bulls with different fertility (Flint et al., 2003). Thus, according to these authors, using heterospermic semen could achieve a higher fertility index than if they used only the semen of that bull with lower fertility. However, we believe that if there are bulls with low fertility, they should not be used for breeding. Therefore, it seems to us that the best economic alternative would 6

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