The buffalo is an important livestock resource in several countries of South Asia and the Mediterranean regions (Warriach et al., 2015; Al-Hamedawi et al., 2017). Puberty is described as the stage in which both males and females become capable of reproducing successfully. It involves various changes in morphology, behavior, and physiology, specifically in the primary reproductive organs (such as the ovaries or testes), as they transition from an immature to a fully developed state (Adams et al., 1994; Al-Mutar et al., 2018).Trans-rectal ultrasonography uses with real time permits precise observing the actions of ovary, particularly measuring the distance of the follicles, rate growth of follicles and ovulation time (Roelofs et al., 2005; Hsan et al., 2019). In numerous studies, ultrasonography has been used to describe changes that occur in the reproductive tract of cows and heifers throughout the estrous cycle (Pierson and Ginther, 1984; Savio et al., 1988; Sirois and Fortune, 1988; Adams, 1999; Sultan et al., 2005). However, our understanding of the pre-pubertal development of the reproductive tract in heifers is limited and derives largely from infrequent (i.e. once a month) examinations of tissues collected at slaughter (Erickson, 1966; Desjardins and Hafs, 1969) or more recent ultrasonographic studies that have focused on ovarian follicular dynamics (Adams et al., 1994; Bergfeld et al., 1994; Evans et al., 1994 a,b; Ginther et al., 1996; Melvin et al., 1999). It has been observed that the levels of circulating estradiol gradually increase as puberty approaches, which is a logical physiological response considering the growing size of ovarian follicles (Evans et al., 1994; Melvin et al., 1999). Progesterone also plays a crucial role in the completion of puberty (Ahmadzadeh et al., 2011; Rahawy and AL-Mutar, 2021). Therefore, the present study aimed for determined the effect of puberty on the follicular size and changes in concentration of progesterone and estrogen in buffalo.KISS1 neurons in the hypothalamus play critical roles in reproductive development and function, including brain-level sex differentiation, puberty onset, and neuroendocrine regulation of gonadotropin production and ovulation (Caraty et al., 2010).

This study was conducted in Sharqat city (Salah Alden Province) during the period from April 2022 to January 2023. This experiment included 18 Iraqi river heifers buffalos divided to three groups (first group which involved six heifers at age of 12 months, second group involved six buffalos aged 18 months and third group involved six buffalos aged 22 months). Estrus detection was done through visual observation in the early morning and evening (Estrus signs were monitored constantly for each females two time a day 07:00 Am and 06:00 Pm for 30 minutes) (Jainudeen, 1988). Ovarian follicular diameters determined using Linear – array intra -rectal transducer of real time B mode with 7.5 MHz (BMD-3000VET, BIOMED, USA). Ultrasound examination of the ovaries was done biweekly from the beginning of the present study and continuously to the first estrus (pubertal age). Blood samples were collected and allowed to clot for 20 minute at room temperature, after that they kept in refrigerator at 5°C for 24 hrs. Serum decanted following immediate centrifugation of the samples (3000 cycle per 15 minutes) and kept under -20°C till analyzed. Hormonal estimation of Progesterone and estrogen were done using ELIZA special kit for each groups monthly till puberty. Statistical analysis of data was performed using SAS (Statistical Analysis System - version 9.1). One-way ANOVA and Least significant differences (LSD) post hoc test were performed to assess significant differences among means (Al-Gharban et al., 2017).

Follicular Size

The results of the clinical examination by the ultrasound device, as shown in Table 1, showed that the largest diameter of the follicle was in the third and second group (12.41 ± 0.17 mm and 11.37 ± 0.30 mm, respectively), with a significant difference P< 0.05 compared to the first group (1.37 ± 0.38 mm).

Table 1: Follicular size during pre, at and post puberty in Iraqi buffalo

Different small letters within Column means significant differences in (P> 0.05)

The results of this study showed that the follicle size was close to what was obtained previously by Honparkhe et al. (2014) in cycling murah water buffalo (11.7 mm); and agree with Awasthi et al. (2006) in Mehsana buffaloes breed (12.9 mm) and Karen and Darwish (2010) in Egyptian buffalo (11.37 mm). The results of these studies were disagreed with Abulaiti et al. (2022) who found that the maximum diameter of dominant follicle (DF) in pubertal and sexually mature crossbred buffaloes was 9.6 ± 2.0 mm and 10.6 ± 0.5 mm, respectively (Sirois et al., 1988). The results of present study were lower than previously obtained by Singh et al. (2020) in the cycling Murah buffalo (14.31 mm). The reason for this difference may be due to many factors: season, nutrition, breed, and environment. Also, these results of follicle size were less than in cows. Ty et al. (1989) recorded that the average size of mature follicles in cows reach 16 mm. Echternkamp et al. (2009) recorded that the follicles diameter in cattle ranging between 14.0 to 17.9 mm, also Luiz et al. (2012) found that the follicles

diameter in the cow ranging from 13-15 mm. The main conclusion of this difference in follicular size in buffalo compared with cows, while the developmental processes are similar may attribute to the number of antral follicles found in buffalo ovaries is only 20% of that of cow ovaries and the number of non atretic follicles (>1.7 mm) average 2.9 for buffalo and 22.1 for cattle (Ty et al., 1989). Dobson and Kamonpatana (1986) found in an abattoir survey, that the proportion of ovaries devoid of surface follicles was much higher in buffaloes than in cattle. Possible reasons for the poor follicular population of buffaloes are a low exit of the reserve of primordial follicles and/or a reduced growth rate of the growing follicles and/or a high extent of atresia (Mauleon and Mariana, 1977; Osamah et al., 2018). As it was demonstrated that neither growth rate nor atresia differed between cows and buffaloes, it is likely that the limited number of growing follicles in buffaloes is linked to a limited initiation of follicular growth from the primordial reserve. The factors causing this require further investigation. They could be either the small size of the reserve of primordial follicles, a feature which is associated in mares with poor folliculogenesis (Driancourt et al., 1982), or a small percentage of easily mobilizable primordial follicles amongst the reserve (Mariana, 1978). In buffalo there are several factors influence the follicular growth such as progesterone levels (Sirois and Fortune, 1988; Knopf et al., 1989) and season (Savio et al., 1990; Badinga et al., 1994). In the pre-pubertal period progesterone seems to be necessary to sensitize ovaries to LH activity in puberty (Roche and Boland, 1991; Fortune, 1993; AL-Sariy et al., 2020).

Estrogen and progesterone

The 17 b-oestradiol causes an increasing in LH secretion in pre-puberty, but the ovulation only occurs prior to progesterone treatment (Badinga et al., 1992). The maximum diameter of the dominant follicle increases near puberty because the increased secretion of 17 boestradiol by these follicles results in the decrease of negative feedback of 17b-oestradiol on LH secretion before puberty (Bergfeld et al., 1994). This mechanism seems to be responsible for the increase in LH secretion (Schillo et al., 1982; Day et al., 1984). Some authors believe this increase in LH secretion is a critical signal involved in the time of puberty in heifers (Madgwick et al., 2005; Al-Hamedawi and Hatif, 2020 a).The results of the current study, as shown in Table 2, showed that the level of estrogen reached its highest level in the second group (at the age of puberty) (30.133 ± 0.87 pg/ml) compared to the first group (before the age of puberty) (22.107±1.52 pg/ml) and the third group, post puberty (18.469 ± 1.08 pg/ml).Same result was recorded by Roy and Prakash (2008) . which recorded This result was agree with other studies (Moran et al. 1989 ; Al-Hamedawi and Hatif, 2020 b) which reported that the estradiol-17 fl is the steroids hormone of most importance for reproduction in heifers. Levels of it remain very low and constant relative to adult patterns, until first ovulation is imminent. This implies that, although they may have important roles in the onset of puberty, neither steroid initiates the process. These increased in estrogen at pubertal age may attribute to an increase of GnRH from mature hypothalamus. Khan et al. (2022) reported that the GnRH acts on the pituitary gland to stimulate the synthesis and release of FSH and LH. Both hormones are responsible for inducing gametogenesis and pubertal maturation. Furthermore, these hormones target the ovaries and testes to produce sex steroids such as estradiol, progesterone, and testosterone, which promote gonadal maturation and are involved in the functional differentiation of the gonads (Cao et al., 2019). Oestrogens play a key role in the regulation of the endocrine and behavioral events associated with oestrous cycle (Roy and Prakash, 2008; Haddawi et al., 2018). The level of progesterone was significantly high P>0.05 in the second and third groups (1.02 ± 033 and 1.43 ± 0.16 ng/ml, respectively) compared to the first group (0.33 ± 0.05 ng/ml), the low plasma progesterone concentrations throughout the growing period before puberty in the present study is quite comparable to that reported by others (Jain and Pandey, 1985; Salama et al., 1994; Alyasiri, 2021). The established statistically significant difference (P<0.01) between progesterone concentrations in the three groups was in line with other research reports (Jain and Pandey,1983; Zaabel et al., 1994; Singh and Madan, 2000; Terzano et al., 2007), affirming that non-cycling animals had low blood concentrations of the studied hormone while those having exhibited ovarian activity had higher blood progesterone values. Puberty onset was associated with the rise in progesterone concentrations (above 1.0 ng/ml for three consecutive blood samples collected every 3-day interval). In Mediterranean Italian buffalo heifers, Terzano et al. (2007) measured average blood progesterone of 0.42 ± 0.19 ng/ml prior to puberty onset.

Table 2: levels of estrogen and progesterone in pre-puberty, at-puberty and post puberty in Iraqi buffalo

Different small letters within Column means significant differences in (P> 0.05)

Effects of combinative genotypes on Puberty and hormonal effect

Association analysis of kisspeptin gene polymorphism was identified using the PCR-RFLP method, the PCR fragments CC, TT and CT genotypes. (Figure 4, Table 3).

Figure 4: The electrophoresis (3 %) showing patterns obtained after digestion with SacI. Note: Fragments including 61 bp of CC and CT genotypes were invisible

Table 3: Shows the association between genotypes and puberty, follicle diameter, estrogen and progesterone

Note: estrogen was significant (P<0.05) whereas the association was not significant for other parameters

Both C\T and C\C SNP were identified in buffalo KISS1 gene, and the c. 374 C>T variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype (Silveira et al., 2010).

The follicular size is reach large size at and post puberty in buffalo with highest diameter in pot puberty. There are no significant differences in progesterone concentration after the heifers reached puberty while the estrogen concentration was decreased as puberty preceding. Three KISS1 SNPs were identified in puberty.

Acknowledgment

The authors are thankful to the faculty members at the Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad, Iraq.

Novelty Statement

The novelty of the study is its focus on the relationship between the age of puberty in Iraqi buffalo with body weight and some biochemical parameters..

Authors’ contributions

These authors each contributed equally.

Conflict of interest

The authors declare that they have no competing interests.

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