Relationship between the leptin, progesterone, body weight, and onset of puberty in ewe lambs

1. Measuring the level of leptin hormone during the pre-pubertal period every two weeks periodically with measuring body weight until it reaches puberty in local ewe lambs.
2. The relationship of leptin and progesterone hormone with the diameter of developing follicles using ultrasound during pre-pubertal and sexual puberty in local ewe lambs.
3. Measuring the diameter of the follicle using an ultrasound device during the pre-pubertal period until it reaches puberty and the appearance of signs of estrus in local ewe lambs.

Introduction

The appearance of puberty in the female sheep depends on both age at first estrus behavior and accumulation of adipose tissues in muscles of body mass (1). Therefore, ewe lamb’s puberty starts when they reach 55-70% of the bodyweight of mature ewe (2). Recently the ‘body weight has been related to puberty and enhanced fertility in ewe lambs (3).Insulin-like growth factor 1(IGF‐1), insulin, growth hormone (GH), follistatin, and leptin hormone play a vital influence role in the accumulation of adipose tissues (4). Leptin is released from adipose tissue, which is a protein in nature with molecular weight 16-kDa composed of 167-amino acid made (5). The importance of leptin hormone play as cellular signaling to regulate the energy metabolisms, body mass, and reproduction activity, Leptin level in ewes is correlate highly with body mass. Therefore, its level decreases during fasting or feed restrictions (6). The occurrence of puberty was found to be linked with "body fat mass which considered as a biomarker for puberty at a critical point of the body mass in ewes (7). Accumulation of adipose tissues and leptin secretion is associated with the onset of puberty and changes in the blood circulating of leptin (2). A different physiological change happens at the beginning of puberty in estrus cycle (8). Monitoring the reproductive system in ewe lambs using ultrasound with trans rectal probes 5.5-7.5MHz is necessary for evaluating ovarian status in terms of anatomical and functional aspects (9). Rectal ultrasonography is used to measure the diameter of the antral follicle and growing follicles with 1-2 mm (10). While some publishers showed a rise in the number of small follicles diameter with 2- is considered as onset of puberty (11).

So, the study aims to determine the relationships between the onset of puberty and body weight with circulating concentrations of leptin and progesterone in prepubertal until the onset of puberty in ewe lambs.

Material and methods

Animals

The study was conducted on the farm of the College of Agriculture, the University of Mosul, eight ewe lamb’s health weaned of local breed at the age of 90 days were used. The project was started in January 2021. They were mixed with sexually male lambs and were kept under the same breeding conditions of light, temperature, and nutrition. Body weights were recorded by electric balance every two weeks periodically from 90 days until the first estrus was detected.

Blood sampling and Hormonal Assay

Samples were collected from the jugular vein and placed in plain sterile tubes (gel tubes). Every two weeks and continuously from the age of 90 days. Blood serum was obtained by separating the blood by a centrifuge for 10 min (3000 rpm) and placed in an Eppendorf tube and stored at -20°C until assays were performed (12).

Sheep leptin assay was estimated by radioimmunoassay (MY BioSource, Competitive ELISA, Antibody-Protein ELISA kit, catalog number: MBS742106, Southern California, San Diego, USA) in duplicate 100 L, as described by Blache et al. (13). The limit value for detection of leptin was 0.06 ng/mL and the intra-assay CVs were 7.3% at0.73 ng/mL, 4.4% at 0.84 ng/ml, and 2.4% at 1.61 ng/mL. Progesterone concentrations were determined by direct solid-phase radioimmunoassay (RIA) (Elecsys Progesterone III; Cobas e411, Roche Diagnostics GmbH, D-68298 Mannheim, Germany) (14).

Ultrasound examination

The study was carried out on local ewe lambs at age weaning, scanning ovaries and follicles periodically every two weeks until the appearance of signs of the first estrus, which is considered signs of sexual puberty. An ultrasound device of the type xianfeng XF30B was used to check the ovarian status in measuring the diameter of the ovaries and the presence of follicles via using transrectal probe 5.5-7.5 MHz with dimensions of 10 cm length, 1.5 cm height, and 1.5 cm width. The probe was inserted into the rectum carefully, about 5-7 cm, where the urinary bladder appeared well, which is the point of access to the animal’s ovary. Once identifying the bladder, the probe was slowly inserted, directing the wave beam at the ventral direction and rotated sideways about 45˚ degrees in both directions until the appearance of the ovary and follicle.

Statistical Analysis

Statistical analyses of the data were evaluated using the statistical SPSS program v.23 software (SPSS In. Chicago, IL., USA). All results were expressed as mean ± standard error (mean ± SE). One-way ANOVA and LSD test evaluated the significance between groups, and P values of less than 0.05 were considered significant (15,16).

Results

As shown in Table 1, the average age of puberty onset significantly differs between days. The earliest estrus behavior was recorded at 202 days (≈6.73 months). The body weight data revealed significant variations among the days at P<0.05, represented by increased body weight. Moreover, the data of leptin level analysis recorded a significant elevation coinciding with the age of puberty, directly and straightforwardly among the studied days at P

Additionally, measuring the follicle diameter by ultrasonography, it was observed that there was an apparent increase in the diameter of the follicle from day 160 until the end of the examination period. It is closely related to the age of puberty (Table 1). (Figures 1 and 2).

Table 1: Effect of time on some physiological parameters associated with pre-puberty in lambs

Different letters in the same column mean significant differences at P<0.05.

Figure 1: Ultrasonographic images show the ovary of ewe lambs with the increase in diameter of the follicle at pre-puberty and puberty days (A, B) at day 160 (C, D) at day 174 (E, F) at day 188.

Figure 2: Ultrasonographic images show the ovary of ewe lambs with the increase in diameter of the follicle at puberty days (G, H, I, J, K, and L) at day 202.

The significant increase of leptin, progesterone, and follicle diameter was positively correlated with the age of puberty and the animal's total weight and pre-puberty days (r= 0.777, 0.814, and 0.764), and (r= 0.909, 0.835, 0.946, and 0.905) respectively. In addition, a close direct relationship was observed between the progesterone hormone level and the follicle diameter with the level of the leptin hormone with the advancing age of puberty (r = 0.742, and 0.682) and the increase in the diameter of the follicle with a rise in the level of serum progesterone hormone (r = 0.956) at the level of probability 0.001 (Table 2).

Table 2: Correlation among the studied parameters

Discussion

The observed and recorded data in the current work were consistent with Lozano et al. (17), who found that the onset of puberty and prevalence of estrus behavior started at 5.4 to 6.9 months. In addition to that, the bodyweight data agreed with Ehtesham and Vakili, Nieto et al. (18,19) that recorded an increase in body weight at the time of puberty 210-240 days, reaching 35 Kg.

Body mass plays a crucial role via changes in growth hormone during aging, especially at the early stages of life till puberty, with the consequence to fat deposition in the body. Fat deposition plays a vital role in puberty and the onset of estrus via increased leptin formation and its release in the body (20). This is logically accepted and supports our finding, which records an increase in leptin level as age progresses and increases body weight (21,22). Therefore, body weight strongly correlates with reproductive health system function and activity in terms of interference with sexual hormones.

This result agrees with Ehtesham and Vakili (18) revealed a positive correlation between increased leptine hormone associated with higher body fat, accumulation of fatty tissue, and increased growth, conversely the increased leptin hormone release (3). Although its role in critical leptin hormone in the early post-natal period processes affects the body and regulation of puberty (21). Leptin hormone concentration was related to age at first estrus and positively correlated with earlier puberty onset was depending on the time of ovarian activity and BW at first estrus in ewe lambs (3).

The present findings found that the progesterone hormone level was significantly increased in age progress toward puberty in ewe lambs. This result agrees with Corner et al. (23), who revealed that during pregnancy, the ewe lambs are not only supporting a developing fetus but are also likely to be still growing themselves. Da Silva et al. (24) have previously shown that lamb’s ewe can partition available resources towards themselves rather than the growing fetus. Progesterone hormone can be used detect puberty in post-weaning ewe lambs. In addition, the progesterone level was more significant in high BW lambs than low BW in ewe lambs. The release of progesterone hormone played an essential role in the prepubertal period that leads to early puberty onset in ewe lambs (25).

Conclusion

The study concludes that puberty in ewe lambs will be early onset with higher breeding values and influenced by an increase in the body weight, concentration of leptin, and progesterone hormone—the positive relationship between them. High leptin and progesterone concentrations were recorded to increase adipose tissue accumulation and muscle, so we must inspect sound body weight effects in puberty in ewe lambs.

Acknowledgment

The authors are very grateful to the University of Mosul, College of Veterinary Medicine, and College of Agriculture for their provided facilities, which helped improve this work's quality.

Conflict of interest

The author has no conflict of interest.