Puberty and parturition on the antral follicle count of female Nelore beef cattle

Puberdade e parto na contagem de folículos antrais de fêmeas Nelore de corte

Abstract

The objective of this work was to investigate whether puberty and parturition alter the antral follicle count (AFC) in female Nelore (Bos indicus) beef cattle. Two experiments were carried out to compare AFC between the prepubertal and pubertal periods and between the prepubertal and postpartum periods. AFC and follicle concentrations did not differ between the prepubertal and pubertal periods. However, the ovarian area increased after puberty. In addition, AFC and the ovarian area were greater in the postpartum than in the prepubertal period. Therefore, AFC is not affected by the puberty of Nelore heifers, but increases significantly when the prepubertal heifers become cows.

Index terms
Bos indicus; artificial insemination; ovulation; pregnancy

Resumo

O objetivo deste trabalho foi avaliar se a puberdade e o parto alteram a contagem de folículos antrais (CFA) em fêmeas Nelore (Bos indicus) de corte. Foram realizados dois experimentos para comparar a CFA entre os períodos pré-púbere e púbere e entre os períodos pré-púbere e pós-parto. A CFA e a concentração folicular não diferiram entre os períodos pré-púbere e púbere. Entretanto, a área ovariana foi maior após a puberdade. Além disso, a CFA e a área do ovário foram maiores no período pós-parto quando comparado ao pré-púbere. Portanto, a CFA não é afetada pela puberdade de novilhas Nelore, mas aumenta significativamente quando as novilhas pré-púberes se tornam vacas.

Termos para indexação
Bos indicus; inseminação artificial; ovulação; prenhez

The development of ovarian follicles in cattle occurs in a successive wave pattern beginning with the simultaneous recruitment of a group of follicles of 1.0-3.0 mm in diameter, followed by the selection of a single follicle and the regression of the others, the subordinate follicles (Ginther et al., 1989). The antral follicle count (AFC) in cattle ovaries is a commonly used phenotypic marker of fertility (Ireland et al., 2011; Mossa et al., 2012; Silva-Santos et al., 2014; Santos et al., 2016), allowing of the prediction of productive and reproductive longevity (Ireland et al., 2008; Jimenez-Krassel et al., 2017), as well as of growth and age at puberty (Santa Cruz et al., 2018). Therefore, depending on its classification, AFC is becoming an optimal target of reproductive biotechnologies (Silva-Santos et al., 2014). Ireland et al. (2008) added that antral follicles ≥ 3.0 mm, observed under a single ultrasonography, can be an important marker for the early selection of female cattle.

The transition of female cattle from prepuberty to full reproductive development requires several body structure and hormonal changes to allow of ovulation, pregnancy, corpus luteum generation, and pregnancy maintenance until parturition. Although studies have already shown that AFC is highly repeatable in the same individual (Burns et al., 2005), there are no known studies on whether AFC remains constant during the different stages of the reproductive life of a same individual.

The objective of this work was to investigate whether puberty and parturition alter the AFC in female Nelore beef cattle.

All experimental procedures were approved by the ethics committee for animal use of Embrapa Rondônia, under protocol number 03/2017.

Two experiments were carried out to compare the AFC between the prepubertal and pubertal periods and between the prepubertal and postpartum periods.

The first experiment was conducted in the municipality of Presidente Médici, in the state of Rondônia, Brazil. Twenty prepubertal Nelore heifers (B. indicus, n=20), weighing 265.79±9.8 kg and with 20±1.3 months of age, were used. The heifers were kept on an Urochloa brizantha (A.Rich.) R.D.Webster pasture with free access to water and mineral supplementation. On a random day of the estrous cycle, the ovaries of the heifers were examined twice in an 11-day interval through transrectal ultrasonography using the A5V 7 MHz linear transducer (Sonoscape, Shenzhen, China). Since there was no corpus luteum in the ovaries, these females were classified as prepubertal. During the initial ultrasonographic evaluation of the ovaries, AFC, and ovarian area (cm²) were recorded. Additional ultrasonography exams were performed monthly until all heifers developed a corpus luteum, which was defined as puberty. Once puberty was reached, the female was examined again to determine AFC and ovarian area. The antral follicles were counted as described in Burns et al. (2005) and Ireland et al. (2008). The total area of the ovary included parenchyma, follicles, and corpus luteum. In the prepubertal and the pubertal periods, the antral follicular concentration per ovarian area was determined by dividing the total AFC by the total area (cm²) of both ovaries, using the following equation: follicular concentration = AFC / ovary area.

The second experiment was carried out in the experimental farm of Embrapa Rondônia, in the municipality of Porto Velho, in the state of Rondônia, Brazil. Thirty prepubertal Nelore heifers (B. indicus, n=30), weighing 261.3±5.8 kg and with 6±0.7 months of age, were initially used. The heifers were kept on a U. brizantha pasture and had free access to water and mineral supplementation throughout the experimental period. The ovaries of the heifers were examined by transrectal ultrasonography using the M5 6 MHz linear transducer (Mindray, Shenzhen, China), in order to evaluate AFC, measure the ovarian area, and determine if all females were prepubertal as defined in the previous experiment. When the heifers reached a live weight of 300 kg, they were subjected to timed artificial insemination (TAI), using the CIDR intravaginal progesterone-releasing device (Zoetis, São Paulo, SP, Brazil) for eight days and 2.0 mg estradiol benzoate (Gonadiol, Zoetis, São Paulo, SP, Brazil) administered via intramuscular injection. Eight days later, the CIDR device was removed and 0.6 mg estradiol cypionate (ECP, Zoetis, São Paulo, SP, Brazil), 150 μg D-cloprostenol (Croniben, Biogenesis Bagó, Buenos Aires, Argentina), and 200 IU eCG (Novormon, Zoetis, São Paulo, SP, Brazil) were administered via intramuscular injection. Two days after the removal of the progesterone-releasing device, all heifers were inseminated using semen from a single bull with proven fertility. Thirty-five days after TAI, ultrasonography was used to detect pregnancies. The heifers that were not pregnant were inseminated again using the same TAI protocol and semen from the same bull. Only the pregnant and calved heifers were included in the study, i.e., 25 of the 30 initially used, of which 18 were pregnant after the first TAI and 7, after the second.

Approximately 50 days after parturition (50±8.5 days), additional ultrasonographic examinations were performed to record AFC and measure ovarian area. Ovarian follicular concentration was calculated in the prepubertal and postpartum periods as described in the first experiment.

AFC, ovarian area, and follicular concentration data were normally distributed according to Shapiro-Wilk’s test. Homoscedasticity and independence of residuals were checked using Levene’s and Durbin-Watson’s tests, respectively. Since regression was observed, linearity was checked using a plot of residual vs. fitted values. All the assumptions were met. The data were then analyzed using a paired data design in a linear mixed-effect model. Pairwise comparisons were performed using the corrected least-squares means. To evaluate the association between AFC and ovarian area, the linear regression analysis was performed, for which the statistical model was selected according to the significance of the coefficient of determination. A significance level of 0.05 was assumed.

In the first experiment, pubertal heifers had a larger ovarian area (p=0.0001) than the prepubertal ones (Figure 1 A), which was expected due to an increased ovarian activity and the presence of corpus luteum after the first ovulation. This result is in agreement with those obtained by Silva-Santos et al. (2014) when evaluating crossbreds (Bos taurus × B. indicus) at weaning from 9 to 24 months of age. The authors classified the heifers into groups with low, medium, and high AFCs, which remained stable throughout the evaluations, confirming that ovarian morphological changes during puberty do not result in an increased AFC. These findings suggest that the AFC in young females is similar before and after puberty.

Figure 1
Ovarian area (A) and follicular concentration (B) in heifers in the prepubertal and pubertal periods in the first experiment and ovarian area (C) and follicular concentration (D) in cows in the prepubertal and postpartum periods in the second experiment. AFC, antral follicle count.

In the second experiment, the ovarian area of the cows was 76% larger after parturition (p=0.0001) in comparison with prepuberty (Figure 1 C). Likewise, Monteiro et al. (2008), in slaughterhouses, found that the ovaries of cows were larger than those of heifers. Contrastingly, Fernandez et al. (2020) did not observe any differences in the size of the ovaries of females aged 12 to 36 months. Ireland et al. (2011) reported a positive relationship between AFC and ovarian size, as well as number of follicles and oocytes in the ovaries of young adult females. In another study, the heifers classified in the low AFC group had 60% smaller ovaries and a much lower total number of follicles and oocytes than those in the high AFC group (Ireland et al., 2008). In the present study, no reproductive status effect was observed on antral follicular concentrations in both experiments (p>0.05) (Figure 1 B and D).

In the first experiment, the AFC of the heifers did not differ significantly (p=0.56) between the prepubertal and pubertal periods, which showed 19.3±1.8 and 20.35±2.3 units of AFC, respectively (Figure 2 A). However, in the second experiment, a higher AFC was observed after parturition (p<0.001) when compared with the prepubertal period, with 36.36±1.8 and 21.67±1.04 units of AFC (Figure 2 B), respectively.

Figure 2
Number of antral follicles in prepubertal heifers that became pubertal (A) and in prepubertal heifers that became cows (B).

Burns et al. (2005) found that AFC varies widely among different females but has a high repeatability in the same individual. In the present study, the same female was compared at different ages (16 months vs. ~34 months), showing an increase of 67% in AFC from the prepubertal to the postpartum period. Unfortunately, the experimental design did not allow of concluding whether this difference between cows and heifers is related to parturition, or to maturity, or to both. In this regard, some studies have shown a positive relationship between AFC and progesterone concentrations in the estrus cycle (Jimenez-Krassel et al., 2009; Ireland et al., 2011). Therefore, it is reasonable to expect that the exposition of the reproductive tract, during pregnancy, to high levels of progesterone for a long period may have some influence on postpartum AFC. The main finding of the present study was that AFC changes dramatically from the prepubertal to the adult phase. Based on these results, prepubertal AFC may not be a good biological marker to predict accurately the AFC of an adult cow as parturition and aging may change its values.

In the first experiment, a positive linear correlation was observed between ovarian area and AFC in the same heifers at prepuberty (p<0.001; R²=0.49) and after puberty (p<0.01, R²=0.5) (Figure 3 A). Similarly, in the second experiment, there was a positive linear correlation between ovarian area and AFC for the same heifers in the prepubertal (p<0.0001, R²=0.54) and postpartum (p<0.01, R²=0.45) periods (Figure 3 B). The obtained results, therefore, indicate that puberty increases the size of the ovaries of Nelore heifers but not their AFC and follicular concentration.

Figure 3
Relationship between ovarian area and number of antral follicles in prepubertal heifers that became pubertal (A) and in prepubertal heifers that became cows (B).

While hormonal, metabolic, and nutritional aspects have been identified as important factors affecting the ovarian follicle population in cattle (Moraes et al., 2019), the relationship between heifer AFC and postpartum timing is poorly understood. Therefore, more comprehensive studies should be conducted to investigate the variation in AFC from the prepubertal to postpartum periods. Although the present work is limited due to the low number of females evaluated and the lack of an extra ultrasound after puberty in the second experiment, the obtained results may be used as a reference for further studies.

Acknowledgments

To Fundação de Amparo à Pesquisa do Estado de Rondônia (FAPERO), for financial support (project 005/2021).

References

• BURNS, D.S; JIMENEZ-KRASSEL, F.; IRELAND, J.L.H., KNIGHT, P.G., IRELAND, J.J. Numbers of antral follicles during follicular waves in cattle: evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations. Biology of Reproduction, v.73, p.54-62, 2005. DOI: https://doi.org/10.1095/biolreprod.104.036277
  » https://doi.org/10.1095/biolreprod.104.036277
• FERNANDEZ, H. de S.; FREITAS, D.C.R. de; GRILLO, G.F.; SILENCIATO, L.N.; CAMARGO FILHO, S.T.; FERREIRA, J.E.; PALHANO, H.B.; MELLO, M.R.B. de. Influence of age and weight of Girolando heifers on uterine and ovarian development. Revista Brasileira de Zootecnia, v.49, e20200028, 2020. DOI: https://doi.org/10.37496/rbz4920200028
  » https://doi.org/10.37496/rbz4920200028
• GINTHER, O.J.; KASTELIC, J.P.; KNOPF, L. Composition and characteristics of follicular waves during the bovine estrous-cycle. Animal Reproduction Science, v.20, p.187-200, 1989. DOI: https://doi.org/10.1016/0378-4320(89)90084-5
  » https://doi.org/10.1016/0378-4320(89)90084-5
• IRELAND, J.L.H.; SCHEETZ, D.; JIMENEZ-KRASSEL, F.; THEMMEN, A.P.N.; WARD, F.; LONERGAN, P.; SMITH, G.W.; PEREZ, G.I.; EVANS, A.C.O.; IRELAND, J.J. Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle. Biology of Reproduction, v.79, p.1219-1225, 2008. DOI: https://doi.org/10.1095/biolreprod.108.071670
  » https://doi.org/10.1095/biolreprod.108.071670
• IRELAND, J.J.; SMITH, G.W.; SCHEETZ, D.; JIMENEZ-KRASSEL, F.; FOLGER, J.K.; IRELAND, J.L.H.; MOSSA, F.; LONERGAN, P.; EVANS, A.C.O. Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of anti-Mullerian hormone as a diagnostic marker for fertility and causes cattle. Biology of Reproduction, v.79, p.1219-1225, 2011. DOI: https://doi.org/10.1071/RD10226
  » https://doi.org/10.1071/RD10226
• JIMENEZ-KRASSEL, F.; FOLGER, J.K.; IRELAND, J.L.H.; SMITH, G.W.; HOU, X.; DAVIS, J.S.; LONERGAN, P.; EVANS, A.C.O.; IRELAND, J.J. Evidence that high variation in ovarian reserves of healthy young adults has a negative impact on the corpus luteum and endometrium during estrous cycles in cattle. Biology of Reproduction, v.80, p.1272-1281, 2009. DOI: https://doi.org/10.1095/biolreprod.108.075093
  » https://doi.org/10.1095/biolreprod.108.075093
• JIMENEZ-KRASSEL, F.; SCHEETZ, D.M.; NEUDER, L.M.; PURSLEY, J.R.; IRELAND, J.J. A single ultrasound determination of≥ 25 follicles≥ 3 mm in diameter in dairy heifers is predictive of a reduced productive herd life. Journal of Dairy Science, v.100, p.5019-5027, 2017. DOI: https://doi.org/10.3168/jds.2016-12277
  » https://doi.org/10.3168/jds.2016-12277
• MONTEIRO, C.M.R.; PERRI, S.H. V.; CARVALHAL, R.; CARVALHO, R.G. Estudo morfológico comparativo dos ovários de vacas e novilhas da raça Nelore (Bos taurus indicus). ARS Veterinária, v.24, p.122-126, 2008. DOI: https://doi.org/10.15361/2175-0106.2008v24n2p122-126
  » https://doi.org/10.15361/2175-0106.2008v24n2p122-126
• MORAES, F.L.Z. de; MOROTTI, F.; COSTA, C.B.; LUNARDELLI, P.A.; SENEDA, M.M. Relationships between antral follicle count, body condition, and pregnancy rates after timed-AI in Bos indicus cattle. Theriogenology, v.136, p.10-14, 2019. DOI: https://doi.org/10.1016/j.theriogenology.2019.06.024
  » https://doi.org/10.1016/j.theriogenology.2019.06.024
• MOSSA, F.; WALSH, S.W.; BUTLER, S.T.; BERRY, D.P.; CARTER, F.; LONERGAN, P.; SMITH, G.W.; IRELAND, J.J.; EVANS, A.C.O. Low numbers of ovarian follicles ≥ 3 mm in diameter are associated with low fertility in dairy cows. Journal of Dairy Science, v.95, p.2355-2361, 2012. DOI: https://doi.org/10.3168/jds.2011-4325
  » https://doi.org/10.3168/jds.2011-4325
• SANTA CRUZ, R.; CUSHMAN, R.A.; VIÑOLES, C. Antral follicular count is a tool that may allow the selection of more precocious Bradford heifers at weaning. Theriogenology, v.119, p.35-42, 2018. DOI: https://doi.org/10.1016/j.theriogenology.2018.06.010
  » https://doi.org/10.1016/j.theriogenology.2018.06.010
• SANTOS, G.M.G. dos; SILVA-SANTOS, K.C.; BARREIROS, T.R.R.; MOROTTI, F.; SANCHES, B.V.; MORAES, F.L.Z. de; BLASCHI, W.; SENEDA, M.M. High numbers of antral follicles are positively associated with in vitro embryo production but not the conception rate for FTAI in Nelore cattle. Animal Reproduction Science, v.165, p.17-21, 2016. DOI: https://doi.org/10.1016/j.anireprosci.2015.11.024
  » https://doi.org/10.1016/j.anireprosci.2015.11.024
• SILVA-SANTOS, K.C.; SANTOS, G.M.G.; KOETZ JÚNIOR, C.; MOROTTI, F.; SILOTO, L.S.; MARCANTONIO, T.N.; URBANO, M.R.; OLIVEIRA, R.L.; LIMA, D.C.M.; SENEDA, M.M. Antral follicle populations and embryo production - In nitro and In vivo - of Bos indicus-taurus donors from weaning to yearling ages. Reproduction in Domestic Animals, v.49, p.228-232, 2014. DOI: https://doi.org/10.1111/rda.12255
  » https://doi.org/10.1111/rda.12255

Publication Dates

• Publication in this collection
  25 Sept 2023
• Date of issue
  2023

History

• Received
  28 Sept 2022
• Accepted
  20 Mar 2023