Relationship between plasma anti-Müllerian hormone concentrations in female Holstein calves immediately after birth and milk yield and composition in dams during early to mid-gestation

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Short Communication Physiology
Abstract: The energy status of dams during the early stages of gestation may affect ovarian reserve.It was hypothesized that greater milk yield and composition during the early stages of gestation may suppress the number of primordial follicles in the fetuses of dairy cattle.This study aimed to evaluate the anti-Müllerian hormone (AMH), used as an indicator of ovarian reserve, in female Holstein calves immediately after birth.It also examined the relationship between the AMH concentrations and the milk yield and composition of dams during early pregnancy.A total of 85 female Holstein calves (born to first-calving cows [nulliparous heifers], n = 31; second-calving [primiparous] cows, n = 22; and third-or subsequent-calving [multiparous] cows, n = 32) were examined.Blood samples were collected from the female calves after birth and before the first colostrum feeding for the analysis of plasma AMH concentrations.Data on daily milk yield and monthly milk composition during the 1st to 6th month of pregnancy were additionally collected from milking dams.The plasma AMH concentrations ranged from 0.106 to 1542.3 pg/mL, with an average of 291.6 ± 30.5 pg/mL.There was no significant difference observed among the plasma AMH concentrations in female calves born from nulliparous heifers, primiparous cows, multiparous cows.Primiparous cows exhibited numerous negative correlations between the plasma AMH concentrations of their female calves immediately after birth and the daily milk yield and milk composition from the 1st to the 4th month of pregnancy; there was, however, no correlation observed in multiparous cows.Overall, there were large individual variations observed in the plasma AMH concentrations immediately after birth.Both milk yield and the amount of milk components strongly influenced the plasma AMH concentrations in calves immediately after birth in primiparous cows in the growth phase.These factors, conversely, did not affect the plasma AMH concentrations of calves in multiparous cows.The results of this study suggest that high-producing pregnant primiparous cows devote more energy to their growth and lactation, potentially compromising the reproductive potential of their offspring.Thus, improving the feeding management of primiparous cows with the goal of increasing the fertility of their progeny will help high-yielding dairy cows reproduce more successfully.
A nti-Müllerian hormone (AMH) is a dimeric glycoprotein of 140 kDa from the transforming growth factor β family (La Marca and Volpe 2006).AMH is primarily secreted by the granulosa cells of healthy growing follicles (La Marca and Volpe 2006) and plays a role in inhibiting primordial follicular growth from the primordial follicle reserve to avoid premature exhaustion of the ovarian follicular reserve (Dewailly et al., 2014).It additionally modulates follicular development by reducing the responsiveness to follicle-stimulating hormones of preantral and small antral follicles (Dewailly et al., 2014).Moreover, AMH is highly positively associated with antral follicle count (Ireland et al., 2008), and therefore, has been used as an indicator of ovarian reserve and potential reproductive longevity (Ireland et al., 2008;Rico et al., 2009).
Follicles are present in bovine fetal ovaries during early stages of gestation, with the number of primordial follicles increasing up to d 110-120 (Erickson 1966;Tanaka et al., 2001).After a peak in the number of these follicles, they either do not markedly change until birth (Tanaka et al., 2001) or decline abruptly toward the end of pregnancy (Erickson, 1966).Mossa et al. (2013) demonstrated that the offspring of dams fed with a 60% restricted energy requirement for maintenance during the first trimester of gestation exhibited lower serum AMH concentrations and antral follicle counts during the growth period than those fed a 120% energy requirement.In other words, a dam's energy level during the early stages of gestation may affect the ovarian reserve, and in turn, the potential reproductive longevity of their offspring.
Primiparous and multiparous Holstein high-producing dairy cows yield approximately 30 and 35 kg of milk per day, respectively (Livestock Improvement Association of Japan, http: / / liaj .lin.gr.jp/japanese/ newmilk/ 22/ R03matome .pdf,2021), during the early stages of gestation, when there is an increase in the number of primordial follicles in the fetus.Furthermore, in addition to allocating ingested nutrients for lactation, primiparous cows also use them for growth, vital maintenance, body fat accumulation, and the placental and fetal development during pregnancy (Redmer et al., 2004;National Agriculture and Food Research Organization, 2017).Previous studies comparing AMH concentrations among

Relationship between plasma anti-Müllerian hormone concentrations in female Holstein calves immediately after birth and milk yield and composition in dams during early to mid-gestation
Saki MORIMATSU, 1 Nagisa NAGAMI, 1 and Chiho KAWASHIMA 1 * parities have shown that primiparous cows have lower AMH concentrations than multiparous cows with 2 and/or 3 parity (Gobikrushanth et al., 2018, Gobikrushanth et al., 2019).Moreover, female Holstein cattle have a lower blood AMH concentrations than female beef cattle (Hirayama et al., 2017;Mossa et al., 2017) and female Jersey cattle (Ribeiro et al., 2014, Gobikrushanth et al., 2019), which produce less milk than Holstein cows.Schwarzmann et al. (2023) found milk yield was lower in multiparous cows with high AMH concentrations than in cows with low AMH concentrations.It was, therefore, hypothesized that one of the reasons high milk producing dairy cows have lower reproductive performance is that higher milk yield, milk components, and growth in primiparous cows during the early stages of gestation may suppress the AMH concentrations of the offspring.However, only a few studies to date have investigated the relationship between the AMH concentrations of the offspring and milk yield of dams.Succu et al. (2020) found that heifers born to dams that were not being milked during gestation had lower AMH concentrations compared with the offspring of cows in their first lactation.Additionally, the antral follicle count of offspring was positively associated with dam milk fat concentration and milk fat-to-protein ratio of dams during pregnancy (Walsh et al., 2014).However, most studies examining blood AMH concentrations were conducted on cattle aged at least 2 mo or older.Mossa et al. (2017) determined that circulating AMH concentrations increased during the first 2 mo of age, decreased at 5 mo of age, and then stabilized at 8 to 9 mo of age around puberty.The study concluded that variations in AMH concentrations observed before puberty reflect changes in the growth patterns of the small antral follicles and alterations in the ability of granulosa cells to secrete AMH.Namely, the nutritional status of the offspring also influences AMH concentrations after birth.Blood AMH concentrations in newborn calves immediately after parturition are, therefore, considered appropriate for assessing the ovarian reserve acquired during the fetal period.However, no studies have investigated the blood AMH concentrations in newborn female calves.Therefore, this study investigated the AMH concentrations in female Holstein calves immediately after birth and before feeding colostrum.It also examined the relationship between AMH concentrations and the milk yield and composition of dams during early pregnancy.
The experimental procedures in the present study were compiled using the Guide for the Care and Use of Agricultural Animals of Obihiro University (approval number: #21-155).A total of 85 female Holstein calves (born to first-calving cows [nulliparous heifers], n = 31; second-calving [primiparous] cows, n = 22; and third-or subsequent-calving [multiparous] cows, n = 32) were born between December 2018 and September 2022.Blood samples of the female calves were collected from the jugular veins immediately after birth and before the first colostrum feeding using sterile 10-mL tubes containing 200 µL of stabilizer solution (0.3 M EDTA, 1% acetylsalicylic acid, pH 7.4) for the analysis of plasma AMH concentrations.The female calves were cleaned, dried with a towel, and weighed before the first colostrum feeding.The tubes were then centrifuged at 2,000 × g for 15 min at 4°C, and the plasma samples were stored at −30°C until analyses.The concentrations of plasma AMH were determined using an enzyme-linked immunosorbent assay kit (Bovine AMH ELISA AL-114; Ansh Labs, Texas, USA).The mean intra-and inter-assay coefficients of variation were found to be 2.6% and 5.8%, respectively.Data on daily milk yield and monthly milk composition were additionally collected during the 1st to 6th month of pregnancy from dams without mastitis (primiparous cows, n = 22; multiparous cows, n = 32).The relationship between plasma AMH concentrations of the female calves and the birth body weight or milk yield and milk composition of their dams were then analyzed using Pearson correlation or Spearman rank correlation analysis after statistical testing for normality using the Shapiro-Wilk test (SigmaPlot® 13; Systat Software, Inc., San Jose, CA, USA.).Moreover, the data were analyzed using a one-way ANOVA (SigmaPlot®) to compare plasma AMH concentrations of female calves among the parity of their dams.The results are reported as the mean ± SEM P < 0.05 was considered statistically significant.
Figure 1 presents a histogram of the plasma AMH concentrations in female calves immediately after birth and before the first colostrum feeding.The plasma AMH concentrations were found to range from 0.106 to 1542.3 pg/mL, with an average value of 291.6 ± 30.5 pg/mL and a median of 194.9 pg/mL.The plasma AMH concentrations of female calves born from nulliparous heifers, primiparous cows, and multiparous cows were found to be 252.0 ± 48.5, 258.8 ± 42.8, and 352.4 ± 58.6 pg/mL, respectively, with no differences among the 3 groups (all, P > 0.1).In addition, although there was a significant difference in birth body weight of the female calves (nulliparous heifers, 41.2 ± 0.7; primiparous cow, 44.4 ± 1.3 and multiparous cow, 44.6 ± 0.9 kg) among the 3 groups (nulliparous heifers vs. primiparous cow, P = 0.007; nulliparous heifers vs. multiparous cow, P = 0.013; primiparous cow vs. multiparous cow, P > 0.1), no correlation was observed between the plasma AMH concentrations and birth body weight (r = −0.034,P= 0.759).
Table 1 presents the coefficient of correlation between plasma AMH concentrations in female calves and milk yield or milk composition of their dams (primiparous cows) during the 1st to 6th month of pregnancy.A negative correlation was observed between plasma AMH concentrations in female calves and the daily milk yield, milk fat content, solid-not-fat content (SNF), milk protein content, and lactose content in their dams in the 1st month of pregnancy.This negative correlation persisted in the 2nd month of pregnancy when the plasma AMH concentrations in female calves were inversely correlated with the daily milk yield, milk fat content, SNF content, milk protein content, and lactose content in their dams.Furthermore, in the 3rd month of pregnancy, plasma AMH concentrations in female calves were negatively correlated with the daily milk yield, SNF content, milk protein content, and lactose content in their dams.Similar negative correlations were found in the 4th month of pregnancy, with the plasma AMH concentrations in female calves being negatively correlated with the daily milk yield, milk fat content, SNF content, milk protein content, and lactose content in their dams.There was, however, no correlation observed between plasma AMH concentrations in female calves and the daily milk yield or milk component amounts in dams in the 5th month of pregnancy.At 6 mo of pregnancy, the only observed negative correlation was found between the plasma AMH concentrations in female calves and the lactose content in their dams.
Table 2 presents the coefficient of correlation between plasma AMH concentrations in female calves and the milk yield or milk composition of their dams (multiparous cows) during the 1st to 6th month of pregnancy.Negative correlations were observed in the 5th month of pregnancy between plasma AMH concentrations in female calves and the milk fat amount and milk protein amount in their dams.At 6 mo of pregnancy, the only observed negative correlation was found between the plasma AMH concentrations in female calves and the daily milk yield in their dams.There was, however, no correlation determined between plasma AMH concentrations in female calves and the daily milk yield or milk components during the other months of pregnancy in their dams.
While there are no reports investigating blood AMH concentrations in female calves immediately after birth, the blood AMH concentrations in Holstein heifers aged 12-15 mo has been reported to range from 53 to 1224 pg/mL (Alward et al., 2021), and from 6 to 440 pg/mL in those aged 11-15 mo (Jimenez-Krassel et al., 2015).Furthermore, the blood AMH concentrations of adult cows during the breeding season, including Holstein, Jersey, and their crossbreeds, ranged from 10 to 3,198 pg/mL (Ribeiro et al., 2014).Postnatal blood AMH concentrations have been demonstrated to increase during the first 2 mo of age, decrease at 5 mo of age, and then stabilize at 8 to 9 mo of age, around puberty (Mossa et al., 2017).Since the concentrations naturally varies according to the month of age, it may not be meaningful to compare the results of the current study with those of earlier research.However, what these studies have in common is that the concentrations range is wide, indicating large individual differences, and the histograms are skewed to the right (a distribution with a short left-hand tail and a large right-hand tail), as in this study.Souza et al. (2015) found strong positive correlations among AMH measurements taken from individual cows at different phases of the estrous cycle and mentioned the high repeatability of AMH measurements across different phases.Additionally, the pregnancy rate in dairy cows with high plasma AMH concentrations is higher than that in dairy cows with low and intermediate concentrations (Ribeiro et al., 2014).Therefore, although the present study did not investigate blood AMH concentrations during the growth period of the female calves examined in this study, this individual difference is likely to be maintained throughout life and reflects the future reproductive performance of the female calves.Primiparous cows, which are still in the growth phase, allocate ingested nutrients not only for lactation, but also for vital maintenance, growth, body fat accumulation, and, during pregnancy, placental and fetal development (Redmer et al., 2004;National Agriculture and Food Research Organization., 2017).In addition, during early pregnancy when milk production is still high, the number of primordial follicles in the fetus increases and reaches maximum on d 110-120 (Erickson 1966;Tanaka et al., 2001).The primiparous cows in this study exhibited numerous negative correlations between the plasma AMH concentrations in their female calves immediately after birth and the daily milk yield and milk component amount from the 1st to the 4th month of pregnancy.This suggests that increased milk and milk component production in primiparous cows requires a higher energy intake, possibly leading to less nutrient distribution for placental and fetal development, thereby resulting in lower plasma AMH concentrations in female calves.Conversely, no correlation was observed between plasma AMH concentrations in multiparous cows and the daily milk yield or milk component amounts during the 1st to 4th month of pregnancy.Akbarinejad et al. (2018) found higher blood AMH concentrations in cows born from multiparous cows compared with those born from nulliparous and primiparous cows.They attributed this difference to differential nutritional partitioning in dairy cows of different parities and the presence of smaller uterine arteries with less blood flow in younger animals with fewer pregnancies than in multiparous animals (Akbarinejad et al., 2018).Moreover, high-producing dairy cows in early pregnancy may benefit from larger individual cotyledons during late pregnancy (Mashimo et al., 2023).This could contribute to providing similar nutrition to the fetus and facilitate the recovery of birth weight compared with low-producing dairy cows (Mashimo et al., 2023).
In this study, there was no significant difference in the plasma AMH concentrations of female calves born from nulliparous heifers or primiparous cow and multiparous cows, despite the high concentrations in the plasma of these calves born from multiparous cows.This study yielded different results from those of Akbarinejad et al. (2018), who investigated blood AMH concentrations when the concentrations was relatively stable following the first calving.The smaller sample size in this study than that in Akbarinejad et al. (2018), and/or the analysis of AMH concentrations immediately after birth, for which there are still insufficient data in this study, could be the cause of the discrepancy in results.This discrepancy, however, could be explained by the fact that in multiparous cows, the placenta and uterine artery development may help supplement the fetuses' nutritional supply and lessen the suppression of primordial follicle proliferation during the early stages of pregnancy (Akbarinejad et al., 2018;Mashimo et al., 2023).In any case, not much is known on AMH concentrations in female calves immediately after birth, and further research is required to address these gaps.Additionally, more research is needed to examine not only milk yield and composition but also factors such as diseases that impact the energy status of the dams to elucidate the factors that affect the number of primordial follicles in fetuses.
In conclusion, our results indicate individual variations in plasma AMH concentrations immediately after birth.Additionally, our findings suggest that both milk yield and milk components exert a strong influence on the plasma AMH concentrations immediately after birth in female calves of primiparous cows in the growth phase, whereas in multiparous cows, no such effect was observed.The results of this study highlight the importance of feeding management in high-producing pregnant primiparous cows, as their allocation of energy to growth and lactation may compromise the reproductive potential of their offspring.Improving the feeding management of primiparous cows to improve the fertility of their offspring may contribute to the successful reproduction of highyielding dairy cows.

Figure 1 .
Figure 1.Histogram of the frequency distribution of plasma AMH concentrations in female Holstein calves immediately after birth and before first colostrum feeding (n = 85).

Table 1 .
MORIMATSU et al. | AMH level in female calves and milk production in dams Coefficient of correlation (r) between plasma AMH concentrations of female calves and milk yield or milk composition of their dams (primiparous cows) during the 1st to 6th month of pregnancy

Table 2 .
Coefficient of correlation (r)between plasma AMH concentrations of female calves and milk yield or milk composition of their dams (multiparous cows) during the 1st to 6th month of pregnancy