THE EFFECT OF OXYTOCIN, PROSTAGLANDIN F2 OR GNRH INJECTION ON FRESH AND FROZEN-THAWED SEMEN CHARACTERISTICS OF RAMS

THE EFFECT OF OXYTOCIN, PROSTAGLANDIN F_2a OR GNRH INJECTION ON FRESH AND FROZEN-THAWED SEMEN CHARACTERISTICS OF RAMS

EL-BADRY D.A.^*; FATEN I. GABR^** and MONA H. SHAKER^*

^*Dept. Artificial Insemination and Embryo Transfer, Animal Reproduction Research Institute, Agriculture Research center

^**Dept. Biology of Reproduction, Animal Reproduction Research Institute, Agriculture Research center

INTRODUCTION

Nowadays increasing use of assisted reproductive techniques in sheep has uncovered a need for additional aids to help veterinarians for optimizing the ejaculates obtained from rams. Specifically, improving upon the number of spermatozoa obtained during semen collection would benefit most areas of assisted reproduction, including semen cryopreservation and artificial insemination. Oxytocin treatment prior to semen collection resulted in increased sperm output in rams (Nicholson et al., 1999; Bozkurt et al., 2007), buffalos(Ibrahim, 1988) bulls (Berndtson and Igboeli, 1988; Palmer et al., 2004) and rabbits (Fjellstrom et al., 1968). Therefore, it seems reasonable that since oxytocin is involved in the transit of sperm through the male duct system, it may hasten ejaculation in rams. Oxytocin may be involved in the movement of sperm in the male duct system, specifically; epididymal sperm transport (Whittington et al., 2001).Oxytocin receptors have been isolated from the epididymal smooth muscle of several species (Frayne and Nicholson, 1998; Whittington et al., 2001) and in vitro and in vivo studies in rodents have shown that oxytocin increased contractility of the epididymis (Hib, 1977). Furthermore, sexual stimulation and ejaculation were associated with a concurrent rise in blood oxytocin concentration in many species (Murphy et al., 1987). 

PG F_2α is known to increase smooth muscle contractility in the male and female genital tracts and may be involved in the ejaculation process(Bygdeman, 1981).  Parenteral PGF_2α administration before semen collection resulted in increased sperm output in stallions (Cornwell et al., 1974; McDonnell, 1992), dogs (Hess, 2001), and buffalo (Ibrahim, 1988). However, the effect of PG F_2α treatment on sperm output in the bull is contradictory, with one study reporting increased sperm output after treatment (Marshall and Hafs, 1976)and a second experiment reporting no treatment effect (Berndtson et al., 1979).

Administration of exogenous GnRH to male dogs induces a dose dependent release of LH immediately following treatment with a resultant increase in testosterone concentration starting 10 minutes after GnRH administration, peak in 50-60 minutes and remains elevated above control values for 4-24 hours (Knol et al., 1993; Hess, 2002). Gabor et al. (1995) found that repeated injection of GnRH resulted in higher level of testosterone hormone in bulls.

The present study attempts to evaluate the effects of oxytocin, PGF2a and GnRH injection prior to semen collection on fresh and frozen-thawed semen characteristics of rams.

MATERIALS and METHODS

Animals and treatment:

Twelve mature, clinically healthy Barki rams, aged 1.5-2.5 years were assigned to the study. Rams were kept in the experimental farm of the Animal Reproduction Research Institute (ARRI), and they were divided into four groups (each containing 3 rams); group 1 (control, injected intramuscularly with 2 ml normal saline 30 minutes prior to semen collection), group 2 (injected with 10 i.u. oxytocin i.v. “Oxytocin, Bela-pharm, Germany”, ^, 10minutes before semen collection  “Knight, 1974”), group 3 (injected i.m. with 250 µg of cloprostenol “Estrumate; Schering Plough Animal Health, Germany” 30 minutes before semen collection “Mekonnen et al., 1989”) and group 4 (injected i.v. with 50 µg of the GnRH analogue, gonadorelin diacetate tetrahydrate “Cystorelin; CEVA Sante Animale, France” ^, 60minutes before semen collection “Hess, 2002”).

Semen collection and evaluation:

Semen samples were collected from rams of all groups using artificial vagina that was adjusted to a proper condition. Two ejaculates were collected once weekly for 10 successive weeks. Immediately after collection samples were evaluated for mass activity and individual motility using a pre-warmed stage of phase contrast microscope. Two smears of the semen, stained with eosin– nigrosin, were prepared (Blom, 1950)and used to determine the percentage of both live and morphologically abnormal spermatozoa (Bielanski et al., 1982).  Sperm concentration was measured with hemocytometer.

Seminal plasma samples:

Seminal plasma samples were obtained at the 1^st, 5^th and 10^th week by centrifugation of portion of semen samples at 3000 rpm for 10 minutes.  Supernatant was passed through 1.2 µm nylon syringe filters to obtain sperm-free seminal plasma and stored at -80 ^oC. Testosterone was measured in seminal plasma samples by ELISA reader in duplicate, at 450 nm wavelength according to Maruyama (1987). The total antioxidant level was measured spectrophotometrically in seminal plasma samples at 37 ^oC and 421 nm according to Koracevic and Koracevic (2001), the superoxide dismutase level was measured at 560 nm according to Nishikimi et al. (1972), while the glutathione peroxidase level was measured at 430 nm according to Paglia and Valentine (1967).

Cryopreservation of ram semen:

Semen samples that collected from all groups at the 1^st, 5^th and 10^th week were diluted at a ratio of 1:19 at 30^oC with Tris based extender (Evans and Maxwell, 1986).After that, the extended semen was cooled to 5^oC throughout 60 minutes in a cold handling cabinet. The cooled semen was loaded into 0.25 ml French straws (IMV, L'Aigle, France), arranged horizontally on a cooled racks, then lowered into liquid nitrogen vapor inside foam box (Khalifa, 2001).Then the straws were immersed in liquid nitrogen and stored.

Evaluation of cryopreserved semen:

One week after cryopreservation, frozen ram semen samples were thawed in water bath at 37^oC for 30 seconds. Individual sperm motility was subjectively assessed post thawing and after 1, 2 and 3 hours of thawing. The post-thawing viability indices were estimated according to Milovanov (1962).The percentage of abnormal acrosome was recorded after thawing in smears stained by fast green FCF (Wells and Awa, 1970).

Statistical analysis:

Two way analysis of variance and Duncan’s multiple range tests were done for the data obtained. Such statistical analysis was performed according toCostat computer program copyright (1986).

RESULTS

As shown in table 1, injection of oxytocin and PGF_2α resulted in a significantly (P ≤ 0.05) higher total semen volume of the 1^st and 2^nd ejaculates (2.84 ± 0.06 and 2.62 ± 0.06 ml, respectively) when compared with the control and GnRH groups (2.17 ± 0.06 and 2.24 ± 0.04 ml, respectively).

Table 1: Effect of injection of oxytocin, PGF2_α and GnRH on semen volume in rams (Mean ± S.D.).

Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Data regarding the ram sperm concentration were represented in table 2.  Injecting rams with PGF_2α prior to semen collection resulted in a significant (P ≤ 0.05) increase in sperm cell concentration when compared to control (2.50 ± 0.04 vs. 2.33 ± 0.05 x10⁹ sperm/ml, respectively).  On the other hand, there were no statistically significant differences (P ≤ 0.05) between oxytocin and GnRH groups (2.29 ± 0.05 and 2.23 ± 0.06 x 10⁹ sperm/ml, respectively) as compared with control group.

Table 2: Effect of injection of oxytocin, PGF2_α and GnRH on sperm concentration in rams (Mean ± S.D.).

  Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Table 3 illustrates the effect of different treatments on the total number of spermatozoa per ejaculates.  Injection of oxytocin and PGF_2α significantly (P ≤ 0.05) increased the total number of spermatozoa per ejaculates as compared with control group (6.59 ± 0.18 and 5.74 ± 0.17 vs. 5.29 ± 0.17 x 10⁹, respectively).  In contrary, the GnRH group had the lowest total number of spermatozoa per ejaculates (4.86 ± 0.14 x 10⁹).

Table 3: Effect of injection of oxytocin, PGF2α and GnRH on total number of spermatozoa per ejaculates in rams (Mean ± S.D.).

   Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Regarding the total number of motile spermatozoa per ejaculates (table 4), the oxytocin group had the highest one (5.15 ± 0.16 x 10⁹) as compared with control, PGF_2α and GnRH groups (4.34 ± 0.14, 4.34 ± 0.12 and 4.14 ± 0.13 x 10⁹, respectively).

Table 4: Effect of injection of oxytocin, PGF2α and GnRH on mass activity of spermatozoa and total number of motile spermatozoa in rams per ejaculates (Mean ± S.D.).

Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

As demonstrated in tables 4 and 5, injection of GnRH resulted in the highest (P ≤ 0.05) mass and individual motility of spermatozoa (+++[+] and 8.33 ± 0.61%).  On the other hand, injection of oxytocin and PGF_2α resulted in a significantly (P ≤ 0.05) lower spermatozoa individual motility as compared with control group (78.17 ± 1.08 and 76.00 ± 0.85% vs. 81.92 ± 0.57%, respectively).

Table 5: Effect of injection of oxytocin, PGF2α and GnRH on Sperm individual motility in rams (Mean ± S.D.).

Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Table 6 illustrates the effect of different treatments on the ram sperm viability.  Injection of GnRH prior to semen collection resulted in the highest (P ≤ 0.05) sperm viability (87.17 ± 0.58%) as compared with control group. On the other hand, injection of oxytocin and PGF_2α resulted in a significantly (P ≤ 0.05) lower sperm viability as compared with control group (80.43 ± 1.04 and 78.30 ± 0.84 vs. 84.08 ± 0.57%, respectively).

Table 6: Effect of injection of oxytocin, PGF2α and GnRH on spermatozoa viability in rams (Mean ± S.D.).

Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Data regarding the percentage of morphologically abnormal spermatozoa are presented in table 7. As compared to control (6.35 ± 0.28%), all the treatment groups resulted in a significantly (P ≤ 0.05) lower percentage of abnormal spermatozoa (5.32 ± 0.24, 5.62 ± 0.31 and 4.52 ± 0.37% for oxytocin, PGF_2α and GnRH groups, respectively).

Table 7: Effect of injection of oxytocin, PGF2α and GnRH on sperm abnormalities in rams (Mean ± S.D.).

   Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

Table 8 illustrates frozen-thawed semen characteristics of rams of different treatment groups. There were no statistically significant (P ≤ 0.05) differences between the control and three treatment groups in the percentage of motile spermatozoa either at 0, 1, 2 or 3 hours post-thawing (just after thawing: 52.22 ± 2.06, 52.78 ± 1.09, 48.89 ± 1.38 and 53.89 ± 1.11% for groups 1-4, respectively; after one hour: 36.67 ± 1.07, 37.78 ± 1.88, 35.00 ± 1.18 and 38.33 ± 1.07% for groups 1-4, respectively; after 2 hours: 25.00 ± 1.07, 25.00 ± 1.44, 24.44 ± 1.30 and 25.56 ± 1.76 for groups 1-4, respectively; at 3^rd hour post-thawing: 15.00 ± 2.89, 13.33 ± 2.04, 14.44 ± 2.26 and 14.44 ± 2.26% for groups 1-4, respectively). Also, there were no statistically significant (P ≤ 0.05) differences between the control and three treatment groups either in viability index (102.78 ± 5.26, 102.50 ± 4.81, 98.33 ± 3.93 and 105.28 ± 2.69 for groups 1-4, respectively) or in the percentage of spermatozoa with acrosomal defects (25.44 ± 1.44, 27.56 ± 1.61, 26.56 ± 1.07 and 26.78 ± 1.34% for groups 1-4, respectively).

Table 8: Frozen-thawed semen characteristics of rams injected with oxytocin, PGF2α and GnRH (Mean ± S.D.).

  Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

As shown in table 9, injection of GnRH prior to semen collection resulted in significant increase in testosterone level when compared with control, oxytocin and PGF_2αgroups (5.16 ± 0.11 vs. 4.34 ± 0.05, 4.37 ± 0.04 and 4.48 ± 0.11, respectively). Concerning the antioxidant activities exerted by ram semen of the different treatments groups (Table 9) represented by total antioxidant, superoxide dismutase and glutathione peroxidase, semen of  PGF_2αgroup has a significantly (P ≤ 0.05) higher antioxidant activities (0.35 ± 0.01, 302.18 ± 5.68 and 18.15 ± 0.92 mM/L, respectively) as compared with control (0.33 ± 0.01, 276.21 ±3.10 and 12.66 ± 0.81 mM/L, respectively), oxytocin group (0.33 ± 0.01, 277.90 ± 3.42 and 13.12 ± 1.11 mM/L, respectively) and GnRH group (0.32 ± 0.00, 224.83 ± 4.52 and 12.66 ± 1.01 mM/L, respectively).

Table 9: The levels of testosterone and antioxidant enzymes in the seminal plasma of rams injected with oxytocin, PGF2α and GnRH (Mean ± S.D.).

    Gp 1: Conrol, Gp2: oxytocin, Gp 3: PGF2α and Gp4: GnRH.

DISCUSSION

In flocks of sheep in which large numbers of spermatozoa must be collected from a few rams over a short period for artificial insemination (A.I.), a substantial increase in the output of spermatozoa per ejaculate per ram would be an advantage (Knight, 1974).The present study aimed to maximize the semen output from certain rams using different regimes as well as to study the effects of these treatments on quality, freezability and antioxidant activity of ram semen.

In the current study, repeated injection of oxytocin resulted in significant increase in semen volume and total number of spermatozoa per ejaculate.  Similar results were recorded for oxytocin in rams (Knight and Lindsay, 1970; Knight, 1974; Voglmayr, 1975; Nicholson et al., 1999; Bozkurt et al., 2007) bulls (Sharma and Hays, 1973; Schefels and ElAzab, 1975; Berndtson and Igboeli, 1988; Palmer et al., 2004), buffalo (Ibrahim, 1988), rabbits (Fjellstrom et al., 1968), dogs (Hess, 2002), men (Rezk et al., 2004) and ostriches (Suttiyotin et al., 2012). In contrast, no detectable effect of oxytocin injection was found in rabbits (Agmo, 1975) and oligospermic men (Byrne et al., 2003). 

In the present work, oxytocin did not alter sperm concentration, sperm mass activity or abnormal sperm rate in comparison to the control values. Similar findings were observed in rams by Bozkurt et al. (2007) and in bulls by Berndtson and Igboeli (1988). Similarly, the addition of oxytocin to bull (Gallagher and Senger, 1989) and stallion semen (Clough et al., 2006) was found to have no effect on sperm motility.

Circulating plasma concentrations of oxytocin increase during sexual arousal and ejaculation in bulls (Sharma and Hays, 1973) and humans (Carmichael    et al., 1987). The action of oxytocin is mediated via activation of the oxytocin receptor  present in the peritubular smooth muscle cells of the epididymis stimulating contractility in vivo (Hib, 1977) and in vitro (Filippi et al., 2002a). Oxytocin acts directly on smooth muscle cells throughout the entire epididymis and indirectly by stimulating the release of endothelin-1 from epithelial cells of the caput epididymidis (Peri et al., 1997; Filippi et al., 2002b). Endothelin-1 is a potent stimulator of epididymal contraction. Epididymal contractility stimulated by oxytocin may be important for the release of sperm stored in the cauda epididymidis, thereby enhancing sperm transport within the extragonadal ducts before and/or during ejaculation (Studdard et al., 2002).

In the current work, repeated injection of PGF_2αresulted in significant increase in semen volume, sperm cell concentration and total number of spermatozoa per ejaculate. Similar results were recorded for PGF_2αin rams (Azawi et al., 2011; Olfati et al., 2013) bulls (Hafs et al., 1974; Haynes et al., 1975; Marshall and Hafs, 1976; Masoumi et al., 2011; Titiroongruang et al., 2011), buffalo (Ibrahim, 1988), stallion (Kreider et al., 1981; McDonnell, 1992), rabbits (Hafs et al., 1974), dogs (Hess, 2002; Kustritz and Hess, 2007) and boars (Hashizume and Niwa, 1984; Estienne and  Harper, 2004). In contrast, it was found that there was no effect of PGF_2αtreatment on various semen characteristics in bulls (Berndtson et al., 1979) and boars (Levis and Reicks, 2005; Kozink, 2002). 

In rams, the addition of PGF_2α to semen increased the conception rate (Gustafsson et al., 1975) and increased the fertility of rams by more than 15%(Dimov and Georgiev, 1977). 

The increase in the sperm number in the ejaculate following Cloprostenol administration is not probably due to an increased rate of spermatogenesis.  Spermatogenesis is unaffected by the collection frequency or short-term PGF_2αadministration (Johnson et al., 1970; Marshall and Hafs, 1976). It has been established that smooth muscle surrounding the epididymis contracts in response to PGF_2αin other species (Hib and Oscar, 1978). Cauda epididymidis acts as a site of storage for mature spermatozoa. When the caudal epididymis contracts in response to PGF_2α, mature spermatozoa are moved into the deferent duct where they are available for ejaculation (Masoumi et al., 2011).In addition to the effects of PGF_2αon the smooth muscle of the epididymis, the testicular capsule also contracts in response to PGF_2α(Free et al., 1980; Cosentino and Cockett, 1986).

In the present study, although GnRH improved motility and viability and morphologically normal spermatozoa of rams, it has no detectable effect on semen volume, sperm cell concentration and total number of spermatozoa per ejaculate. These results coincided with the findings of Hess (2002)in dogs. In contrary, Gabor et al. (1995) observed that repeated injection of GnRH provided valuable results of producing capacity of bull testes. Also, Braun et al. (1988) revealed the improvement of semen quality in adult bulls after the administration of GnRH. Garner et al. (1990) added that GnRH controls the production of gonadotropins, thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis.

In the current investigations, repeated GnRH injection resulted in marked increase in testosterone level as compared to control. These results coincided with that recorded in bulls by Braun et al. (1988) and Gabor et al. (1995) and in dogs by Knol et al. (1993). The data pertaining to the effect of oxytocin on testosterone are controversial, because some researchers (Inaba et al., 1999) have reported that oxytocin treatment reduces the testosterone concentration in goats, whereas others (Nicholson et al., 1987) have reported that this hormone increases the release of testosterone in rats, and some (Nozdrachev et al., 1994; Kumar and Farooq, 1994; Bozkurt et al., 2007 in rat, mice and rams, respectively) have also documented that oxytocin does not alter the concentration of testosterone. In the present study, it was observed that the administration of oxytocin did not affect the levels of seminal plasma testosterone at any time compared to the control group. In the current work, injection of PGF_2α had no effects on the level of testosterone. On contrary, plasma testosterone concentrations in bulls were increased after PGF_2αinjection (Haynes et al., 1975; Kiser et al., 1976; Masoumi et al., 2011).

An unbalanced, excessive production of ROS and decreased level of antioxidant enzymes cause decreased sperm motility and viability, and increased sperm defects by initiating an oxidation chain reaction damaging proteins, lipids and DNA (Sikka, 2004; Aitken and Baker, 2004).Seminal plasma has an antioxidant system that seems to be very relevant to the protection of sperm. The sperm oxidative defence enzymes predominantly include superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase (Sarlos et al., 2002).In the present study, PGF_2α had beneficial effects on the antioxidant activities while repeated injection of oxytocin and GnRH had no deleterious effects on antioxidant activities in ram semen in terms of total antioxidants, superoxide dismutase and glutathione peroxidase levels in seminal plasma. To the best of our knowledge, no previous data are available about the effect of repeated injection of oxytocin, PGF_2α and GnRH on the antioxidant activities of semen.

Concerning the post-thaw semen characteristics in the present work, all the treatment groups did not alter motility, viability and acrosomal integrity of the frozen-thawed ram semen.  In agreement with these results, the others reported that the administration of PGF_2αto bulls prior to ejaculation did not influence the motility of frozen-thawed spermatozoa (Marshall and Hafs, 1976; Masoumi et al., 2011). Berndtson and Igboeli (1988) mentioned that oxytocin did not impair the ability of bull spermatozoa to withstand freezing or thawing.

In conclusion, it was suggested that injection of PGF_2α andoxytocinprior to semen collection may be useful when collecting ram semen for use in artificial insemination leading to increase in the semen output and increment in the total number of straws frozen. Moreover, repeated injection of them neither causes depletion of sperm reserves in rams nor alters the quality of fresh and frozen-thawed semen as well as they have no deleterious effects on the antioxidant activities of seminal plasma and testosterone level in rams.

REFERENCES

Agmo, A.J. (1975): Neurohypophysial hormones and the emission of semen in rabbits. J. Reprod. Fertil., 45, 243-248.

Aitken, R.J. and Baker, M.A. (2004): Oxidative stress and male reproductive biology.  Reprod. Fertil. Dev., 16: 581–588.

Azawi, O.I.; Al-Khasab, A.T. and Al-Kadoo, N.N. (2011):  Study on the effect of prostaglandin F2α treatment on semen characteristics and enzymatic activates of Awassi rams in breeding and non breeding seasons.  Res. Opinions Anim. Vet. Sci., 1 (5): 318-324.

Berndtson, W. and Igboeli, G. (1988): Spermatogenesis, sperm output and seminal quality of Holstein bulls electroejaculated after administration of oxytocin. J. Reprod. Fertil., 82: 467–475.

Berndtson, W.; Chenoweth, P.; Seidel, G.; Pickett, B. and Olar, T. (1979): Influence of prostaglandin F2α on spermatogenesis, spermatozoal output, seminal quality, testosterone levels and libido of yearling beef bulls. J. Anim. Sci., 49:     736–742.

Bielanski, W.; Dudek, E.; Bittmar, A and Kosiniak, K. (1982): Some characteristics of common abnormal forms of spermatozoa in highly fertile stallions. J. Reprod. Fertil. Suppl., 32: 21–26.

Blom, E. (1950): Eine schnellfarb methode mit eosin und nigrosin zur unterscheidung von lebenden und toten spermien. Wien Tierarztl Wschr, 37: 441–442.

Bozkurt, T.; Gaffari, T.R. and Seyfettin, G.R. (2007): Effects of Exogenous Oxytocin on Serologic and Seminal Steroids and Semen Characteristics in Rams. Turk. J. Vet. Anim. Sci.; 31(5): 303-309.

Braun, J.; Brem, G. and Schams, D. (1988): Reproductive functions in monozygotic bulls derived from embryo-microsurgery. In: Proceedings of VI. World Conf. of Anim. Prod. Helsinki., pp: 577-580.

Bygdeman, M. (1981): Effects of prostaglandins on the genital tract. Acta. Vet. Scand., 77 (Suppl.): 47–54.

Byrne, M.M.; Rolf, C.; Depenbusch, M.; Cooper, T.G. and Nieschlag, E. (2003): Lack of effect of a single i.v. dose of oxytocin on sperm output in severely oligozoospermic men.  Human Reprod., 18 (10): 2098-2102.

Carmichael, M.S.; Humbert, R. and Dixen, J. (1987): Plasma oxytocin increases in the human sexual response. J. Clin. Endocrinol. Metab., 64: 27–31.

Clough, C.J.; Campbell, M.L.H. and Matson, T. (2006): The effect of inclusion of oxytocin in semen extender on spermatozoal motility. Anim. Reprod. Sci., 94: 132–134.

Cornwell, J.; Koonce, K. and Kreider, J. (1974): Effect of prostaglandin F2α on seminal characteristics of the stallion. J. Anim. Sci., 38: 226 (Abst.).

Cosentino, M.J. and Cockett, A.T. (1986): Structure and function of the epididymis. Urol. Res., 14: 229-240.

Costat Computer Program copyright (1986): Version3.03 copyright software.

Dimov, V. and Georgiev, G. (1977): Ram semen prostaglandin concentration and its effect on fertility J. Anim. Sci., 44 (6): 1050-1054.

Estienne, M.J. and Harper, A.F. (2004): PGF2α facilitates the training of sexually active boars for semen collection. Theriogenology, 54: 1087-1092.

Evans, G. and Maxwell, W.M.C. (1986): Salamon's Artificial Insemination of Sheep and Goats. Butterworth, Sydney.

Filippi, S.; Luconi, M.; Granchi, S.; Vignozzi, L.; Bettuzzi, S.; Tozzi, P.; Forti, G. and Maggi, M. (2002a): Estrogens, but not androgens, regulate expression and functional activity of oxytocin receptor in rabbit epididymis. Endocrinol., 143: 4271-4280.

Filippi, S.; Vannelli, G.B.; Granchi, S.; Luconi, M.; Crescioli, C.; Mancina, R.; Natali, A.; Brocchi, S.; Vignozzi, L. and Bencini, E. (2002b): Identification, localization and functional activity of oxytocin receptors in epididymis. Mol. Cell. Endocrinol., 193:      89-100.

Fjellstrom, D.; Kihlstrom, J. and Melin, P. (1968): The effect of synthetic oxytocin upon seminal characteristics and sexual behaviour in male rabbits. J. Reprod. Fertil., 17: 207–209.

Frayne, J. and Nicholson, H. (1998): Localization of oxytocin receptors in the human and macaque monkey male reproductive tracts: evidence for a physiological role in the male. Mol. Hum. Reprod., 4: 527–532.

Free, M.J.; Jaffe, R.A. and Morford, D.E. (1980): Sperm transport through the rete testis in anesthetized rats: role of the testicular capsule and effect of gonadotropins and prostaglandins. Biol. Reprod., 22: 1073-1078.

Gabor, G.; Mayes, Z.; Sziicsz, E. and Banary, I. (1995): Relationship among testosterone response to GnRH administration, testes size and sperm parameters in Holstein-Friesian bulls. Theriogenology, 43: 1317-1324.

Gallagher, G.R and Senger, P.L. (1989): Effect of phenylephrine, ergonovine, oxytocin and norephrine as an extender ingredient on viability of bovine spermatozoa. J. Anim. Sci., 67 (6): 1573–1576.

Garner, L.L.; Campbell, G.T. and Blake, C.A. (1990): Luteinizing hormone (LH)-releasing hormone: chronic effects on LH and follicle-stimulating hormone cells and secretion in adult male rats. Endocrinology, 126 (2): 992-1000.

Hafs, H.D.; Louis, T.M.; Waters, R.J.; Stellflug, J.N. and Haynes, N.B. (1974): Increased sperm output of rabbits and bulls treated with PGF2α. Prostaglandins, 8: 417-422.

Hashizume, T. and Niwa, T. (1984): Effect of administration of PGF2α on the properties of sperm rich fraction of boar semen. Japan J. Anim. Reprod., 30: 182-185.

Haynes, N.B.; Hafs, H.D.; Waters, R.J.; Mannas, J.G. and Riley, A. (1975): Stimulatory effect of PGF2α on the plasma concentration of testosterone in bulls. J. Endocrinol., 66:      329-338.

Hess, M. (2001): The effects of prostaglandin-F2_, oxytocin and gonadotropin releasing hormone on ejaculate characteristics in the dog, Proc. Soc. Theriogenology, 15.

Hess, M. (2002): The effects of prostaglandin F2α, oxytocin and gonadotropin releasing hormone on ejaculate characteristics in the dog. Master Thesis, Virginia Polytechnic Institute and State University, USA.

Hib, J. (1977): The “in vivo” effects of oxytocin and vasopressin on spontaneous contractility of the rat epididymis. Int. J. Fertil., 22: 63–64.

Hib, J. and Oscar, P. (1978): Effects of prostaglandin and indomethacin on rat epididymal responses to norepinipherine and acetylcholine. Arch. Androl., 1: 43-47.

Ibrahim, M. (1988): Influence of oxytocin and prostaglandin on semen characteristics and process of ejaculation in buffalo bulls. Acta. Vet. Hung., 82: 3–10.

Inaba, T.; Nakayama, Y.; Tani, H.; Tamada, H.; Kawate, N. and Sawada, T. (1999): Oxytocin gene expression and action in goat testis. Theriogenology, 52: 425-434.

Johnson, A.D.; Gomes, W.R. and Van Demark, N.L. (1970): “The Testis”. Academic Press.

Khalifa, T.A.A. (2001): Effect of some antioxidants on viability of preserved buffalo and ram semen. Ph.D. Thesis, Fac. Vet. Med. Cairo Univ.

Kiser, T.E.; Hafs, H.D. and Oxender, W.D. (1976): Increased blood LH and testosterone after administration of PGF2α in bulls. Prostaglandins, 11: 545-553.

Knight, T.W. and Lindsay, D.R. (1970): Short- and long term effects of oxytocin on quality and quantity of semen from rams. J. Reprod. Fert., 21: 523-529.

Knight, T.W. (1974): The effect of oxytocin and adrenaline on the semen output of rams. J. Reprod. Fertil., 39: 329-336.

Knol, B.W.; Dieleman, S.J. and Bevers, M.M. (1993): GnRH in the male dog: dose response relationships with LH and testosterone. J. Reprod. Fertil., 98: 159.

Koracevic, D. and Koracevic, G. (2001): J. Clin. Path., 54: 356-361.

Kozink, D.M. (2002): Enhancing boar reproductive performance for purposes of artificial insemination. M.V.Sc. Thesis, Georgia Univ., U.S.A.

Kreider, J.L.; Ogg, W.L. and Turner, J.W. (1981): Influence of prostaglandin F2alpha on sperm production and seminal characteristics of the stallion. Prostaglandins, 22: 903–913.

Kumar, A. and Farooq, A. (1994): Effect of oxytocin on the concentration of fructose in the accessory glands of mouse. Life Sciences, 55 (l):19-24.

Kustritz, M.V. and Hess, M. (2007): Effect of administration of prostaglandin F2alpha or presence of an estrous teaser bitch on characteristics of the canine ejaculate. Theriogenology, 67: 255–258.

Levis, D.G. and Reicks, D.L. (2005): Assessment of sexual behavior and effect of semen collection, pen design and sexual stimulation of boars on behavior and sperm output—a review. Theriogenology, 63: 630–642.

Marshall, C. and Hafs, H. (1976): Sperm output and quality after PGF in bulls. J. Anim. Sci., 43, 296 (Abst.).

Maruyama, Y. (1987): Sex-steroid-binding plasma protein, testosterone, oestradiol and DHEA in prepuberty and puberty. Acta Endocronologica, 114: 60-67.

Masoumi, R.; Towhidi, A.; Javaremi, A.N.; Nabizadeh, H. and Zhandi, M. (2011): Influence of PGF2α on semen quality and libido in Holstein bulls. Turk. J. Vet. Anim. Sci., 35 (1): 1-6.

McDonnell, S. (1992): Ejaculation physiology and dysfunction. Vet. Clin. N. Am. Equine Prac., 8: 57–70.

Mekonnen, G.; Boland, M. and Gordon, I. (1989): The effect of prostaglandin on semen production and libido in the ram. Irish Vet. J., 1 (1-2): 56-62.

Milovanov, V.K. (1962): Biology of reproduction and artificial insemination of farm animals. Monograph. Selkohz. Lit. J. and Plakatov, Moscow.

Murphy, M.R.; Seckl, J.R.; Burton, S.; Checkley, S.A. and Lightman, S.L. (1987): Changes in oxytocin and vasopressin secretion during sexual activity in men. J. Clin. Endocr. Metab., 65:738-741.

Nicholson, H.; Parkinson, T. and Lapwood, K. (1999): Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep.  J. Reprod. Fertil., 117: 299–305.

Nicholson, H.D.; Worley, R.T.S.; Guldenaar, S.E.F. and Pickering, B.T. (1987): Ethan-1,2-dimethanesulphanate reduces testicular oxytocin content and seminiferous tubule movements in the rat. J. Endocrinol., 112:  311-316.

Nishikimi, M.; Rao, N.A. and Yogi, K. (1972): Bioch. Biophys. Res. Common., 46: 849-854.

Nozdrachev, A.D.; Kovalenk, R.I.; Chernysheva, M.P. and Semenova, E.P. (1994): The effect of the intraventricular administration of oxytocin on the functional activity of the epiphysis, adrenals and gonads and on the behavior of rats. Fiziol. Zh. I. I.M. Sechenova, 80: 88-97.

Olfati, A.; Moghaddam, G.H.; Daghigh, H. and Shabankareh, H.K. (2013): Effects of prostaglandin F2α treatment on semen characteristics of crossbred rams in the non-breeding season. J. Cell & Anim. Biol., 7 (2): 16-20.

Paglia, D.E. and Valentine, W.N. (1967): J. Lab. Clin. Med., 70: 158-169.

Palmer, C.W.; Amundson, S.D.; Brito, L.F.; Waldner, C.L. and Barth, A.D. (2004): Use of oxytocin and cloprostenol to facilitate semen collection by electroejaculation or transrectal massage in bulls. Anim. Reprod. Sci., 80: 213-223.

Peri, A.; Fantoni, G.; Granchi, S.; Vannelli, G.B.; Barni, T.; Amerini, S.; Pupilli, C.; Barbagli, G.; Forti, G. and Serio, M. (1997): Gene expression of endothelin-1, endothelin-converting enzyme-1, and endothelin receptors in human epididymis. J. Clin. Endocrinol. Metab., 82, 3797-3806.

Rezk, A.Y.; Al-Inany, H. and Bedaiwy, M. (2004): Multiple intravenous oxytocin injection may yield sperm in azoospermic men scheduled for TESE. Middle East Fert. Soc. J., 9: 238-241.

Sarlos, P.; Molnar, A.; Kokai, M. and Ratky, G. (2002): Comparative evaluation of the effect of antioxidants in the conservation of ram semen. Acta Veterinaria Hungarica, 50: 235–245.

Schefels, W. and ElAzab, A. (1975): The effect of oxytocin on ejaculate composition in the bull. Theriogenology, 4: 134, Abstr.

Sharma, O.P. and Hays, R.L. (1973): Release of an oxytocic substance following genital stimulation in bulls. J. Reprod. Fert., 35:    359-362.

Sikka, S.C. (2004): Role of oxidative stress and antioxidants in andrology and assisted reproductive technology.  J. Androl., 25: 5–18.

Studdard, P.W.; Stein, J. L. and Cosentino, M.J. (2002): The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat. Int. J. Androl., 25: 65-71.

Suttiyotin, P.; Khumpim, P.; Kimsakulvech, S. and Eurlaphan, C. (2012): Improvement of semen quantity and quality in Ostriches by oxytocin injection before semen Collection. Suranaree J. Sci. Technol., 19 (1): 9-14.

Titiroongruang, J.; Hirunpattarawong, P.; Sophonpattana, P.; Singlor, J. and Tummaruk, P. (2011): Effects of Prostaglandin F2α on Serum Testosterone and Semen Output in Holstein Friesian Bulls in Tropical Climate. Thai. J. Vet. Med., Suppl. 41:159-160.

Voglmayr, J.K. (1975): Output of spermatozoa and fluid by the testis of the ram and its response to oxytocin. J. Reprod. Fert., 43: 119-122.

Wells, M.E. and Awa, O.A. (1970): New technique for assessing acrosomal characteristics of spermatozoa.  J. Dairy sci., 53: 227-232.

Whittington, K.; Assinder, S.; Parkinson, T.; Lapwood, K. and Nicholson, H. (2001): Function and localization of oxytocin receptors in the reproductive tissues of rams.  Reproduction, 122: 317–325.