Effect of Melatonin Implantation on Sexual Behavior and some of Semen Quality of Iraqi Buffalo Bulls

This study investigated the effect of melatonin implantation on sexual behavior and some aspects of the semen quality of buffalo bulls. Nine buffalo bulls had divided into three groups (3/group). The first group was the control group (C1). The buffalo bulls were implanted with melatonin hormone in the second (72 mg; C2) and third (90 mg; T3) groups under the skin of the left ear base and repeated one month after the first implantation. Semen was collected for 14 weeks and evaluated weekly. The first amount of time was lower in the C3 group than in the C2 and C1 groups. Semen volume at 2nd ejaculation was higher (P<0.05) in the C3 group than in other groups. Mass activity and motility increased in the C3 group compared to the C2 and C1 groups at different ejaculation times. The melatonin implantation (90 mg) can improve the semen quality of Iraqi bulls which in turn enhancing the reproductive performance, conception rate of female buffalo and increasing the artificial insemination productivity and owner’s income.


Introduction
Artificial insemination and efficient progeny testing programs are vital indicators of genetic improvement [1][2][3].Sperm cryopreservation has contributed to reducing the obstacles and factors that make it hard to obtain semen, such as the geographical location and the time factor [4].The semen of buffalo bulls is high in unsaturated fatty acids and contains a few antioxidants [5,6] compared to the Holstein bulls [7,8,9].Therefore, the semen of buffaloes is more susceptible to lipid oxidative damage by free radicles of reactive oxygen species (ROS), which leads to an increase in the percentage of abnormalities, damage to the genetic material, and a decrease in fertility [10,11].In semen, free radicles or ROS are generated by leukocytes, dead, abnormal, and immature spermatozoa.Buffalo bulls are characterized by a lack of sperm concentration, motility, and viability, a weak sperm mitochondrial membrane, and maturation within the epididymis [12].The effects of free radicals or ROS on the semen of buffalo bulls appear during spermatogenesis and cryopreservation [6,11,13,14].The major obstacle facing antioxidant addition to the buffalo bull's semen extender is the failure to treat the affected sperm with reactive oxygen species during the spermatogenesis process in the testes, so there is an urgent need to give antioxidants by treating the animals directly [15].Melatonin hormone is secreted from the pineal gland, and its receptors are found in many cells and organs of the body, which indicates the evident effectiveness of the melatonin hormone, as it regulates the daily and seasonal harmony of vital activities in the body and enhances the immune status of the body [16].It works directly in removing all kinds of free radicals and indirectly as an antioxidant through 1262 (2023) 072013 IOP Publishing doi:10.1088/1755-1315/1262/7/072013 2 the activation of enzyme antioxidants as well as its active role as an anti-inflammatory, and inhibition of oxidative stress enzymes [17,18], which reduces oxidative damage in cells [19].Its protective effect as a powerful antioxidant is due to its high effectiveness in inhibiting fat oxidation by removing free radicals, reactive oxygen, and nitrogen species [20,21].Ramadan [22] indicated that implantation of buffalo bulls with 18 mg/50 kg live weight outside the breeding season led to an improvement in the semen quality of the bulls, total protein concentration, albumin, cholesterol, and the activity of the enzyme superoxide dismutase in the seminal plasma compared to the untreated bulls.Eidan et al. [23,24,25] and Mahmood et al. [17,20] showed that implantation of Holstein bulls with the hormone melatonin (72 and 90 mg/bull) significantly improved some of the semen quality, the total antioxidant capacity, and catalase concentrations compared with the control treatment.Due to the lack of studies on the effect of melatonin implantation in the breeding season of Iraqi buffaloes, this experiment investigated the effect of melatonin implantation on some of the sexual behavior and semen characteristics of fresh buffalo bulls.

Materials and Methods
This study has carried out in the Department of Artificial Insemination of the Department of Animal Resources -Ministry of Agriculture in Abu Ghraib, Baghdad.Nine Iraqi buffalo bulls (5-7 years old and 550-800 kg weighting) were trained for semen collection using an artificial vagina.The experimental buffalo bulls have good health.Bulls had divided into three groups.The first group was considered a control group (C1).The second (C2; 72mg/ bull) and third (C3;90 mg/bull) groups were implanted with melatonin hormone under the skin of the left ear base and repeated one month after the first implantation.Semen was collected using a previously prepared artificial vagina with 41-420 C and teaser.The semen has collected for 12 weeks.The time for the first, second, and third amount and ejaculation were recorded [26].Fresh semen had evaluated regarding the volume, sperm concentration, mass activity, and individual motility [23,24,25,27].

Statistical Analysis
The statistical computation were performed using SAS program based on study on completely randomized deign to study the effect of different factors on the studies characteristics.Yij = µ + Ti + eij Yij = dependent variable pertaining to the j observation of the i treatment.µ = overall mean.Ti = effect of ith treatment (i-C1, C2 and C3 groups).eij = error term.Means with significant differences were compared using Duncan multiple range test.

Results and Discussion
The C3 group recorded less (P˂0.05)first mounting time of the buffalo bulls than the C1 and C2 groups (Table 1).Simultaneously, the C3 group exhibited less (P˂0.01)second mounting time of the buffalo bulls than the C2 groups (Table 1).Moreover, no significant differences among groups at a third and average mounting time (Table 1).Less time for first and second ejaculation had recorded in C3 and C1 groups compared to the C2 group.No significant differences among groups at a third and average ejaculation time (Table 1).The sexual behavior of bulls is an expression of both sexual desire and the ability to breed [28].In the artificial insemination process, it is necessary to maintain a high sexual desire in the bull to benefit from the ejaculation.Bulls with weak sexual desire are unfit for reproductive purposes.The lack of time for mounting and ejaculation in the C3 group for buffalo bulls may be due to the lack of secretion of the adrenal stimulating hormone from the hypothalamus, which leads to a lack of secretion of the hormones ACTH and cortisol, and thus the increased secretion of testosterone [29], which has reflected in the short duration of the response to mounting and ejaculation.The results revealed a significant increase (P˂0.05) in the second ejaculation volume and average volume in the C3 group compared to the C1 groups (Table 2).High concentration of semen recorded in C1 group (1176.50±58.6 ᵡ106) in comparison with C2 (921.18±91.63ᵡ106)and C3 (851.33±47.79ᵡ106) groups for first ejaculation (Table 2).The mass activity was higher in the C3 group than C1 (2nd and average mass activity) and C2 (1st and average mass activity) groups (Table 3).The C3 group showed a significant increase (P˂0.05) in sperm motility compared with the rest of the groups (C1 and C3) for 1st, 2nd, 3rd, and average motility (Table 3).The current data agreed with those reported by Ramadan [22], who noticed an improving semen quality of buffalo bull's implantation melatonin (18mg/50kg body weight) during the nonbreeding season under the tropical condition as compared to a control group.The improvement of semen quality in a group treated with 92 mg melatonin (C3) may be to increase testosterone secretion as discussed in the above paragraph.Another reason for the improvement of some semen quality in buffalo bulls implantation90 mg melatonin hormone may be to increase the level of melatonin in the semen of these bull.The Melatonin hormone plays a key role in testicular steroidogenesis and spermatogenesis [31].It directly regulates androgen and testosterone secretion and increases the responsiveness of Sertoli cells to follicle-stimulating hormones during spermatogenesis.Melatonin may be applied in a range of applications not only to maintain sperm viability but also to modulate sperm metabolism and physiology [31].Melatonin and its metabolites act as free radical scavengers, protecting cells from oxidative stress, and could have a significant impact on infertility.Melatonin is absorbed by the testis.Melatonin affects testicular function directly by acting on receptors in Leydig and Sertoli [32].Awad [33] observed lower melatonin levels in men affected by nonobstructive azoospermia, in those displaying impaired sperm motility.The recent finding was in disagreement with Eidan et al. [23,24,25] results, they didn't find any significant differences between melatonin implantation and non-implantation of Holstein bulls in ejaculation volume, motility, mass activity, and concentration.These inconsistencies with our results may be due to different species or environments.
Table 1.Effect of the melatonin implantation in Iraqi buffalo bulls on sexual behavior (mean ± standard error).

Conclusion
In conclusion, implantation of buffalo bulls with 90 mg melatonin hormone has a positive role in improving the sexual behavior and some semen quality compared to the control group, which in turn enhances the reproductive performance, and conception rate of female buffalo and increases the artificial insemination productivity and owner's income

Table 2 .
Effect of the melatonin implantation in Iraqi buffalo bulls on volume and concentration of semen (mean ± standard error).
Means with different superscripts within similar raw differ significantly; C1 = control group; C2 = buffalo bulls were implanted with 72 mg melatonin hormone/ month for 2 months; C3= buffalo bulls were implanted with 90 mg melatonin hormone/ month for 2 months.

Table 3 .
Effect of the melatonin implantation in Iraqi buffalo bulls on mass activity and motility of semen (mean ± standard error).Means with different superscripts within similar raw differ significantly; C1 = control group; C2 = buffalo bulls were implanted with 72 mg melatonin hormone/ month for 2 months; C3= buffalo bulls were implanted with 90 mg melatonin hormone/ month for 2 months.