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Effects of testosterone injection on the semen quality in boars during high ambient temperature
Animal Repr&c~ionSci‘mce. 25 (1991) 73-E?. Elsevier Science Publishers B.V., Amsterdam
73
Effects of testosterone injection on the semen quality in boars during high ambient temperature
ABSTRACT NPkayama. Ii.. Hidaka. R. and Ashizawa. K.. 1991. Effects of tatosterone injection on the semen quality in boars duting high ambient temperature. Anin?, Rept(h;. Sci, 25: 73-82. Attempts were made to inhibit the reduction in wnen quality and reproductive pcrfoormanceby injecting testoslemne into boars during the hot summer months. Testosterone enanthate (250 cn 500 mg pr boar) was injccled into the rump muscle ofboan once a month from June to September. The most t’cmarirabIecffectr were obtained in rhe incidwtce ofabnormat spermatows. In themnlml group (no injnian). the percentagegel spermatozoa i.e. those with proximal cytoplasmif drop lets. abnommt heads and roiled and bent tails increased to 34.3 f 3.6% in September. In ~ntrasl, the pmntaacs of abnormal spermatozoa in the testostemne-injectedamups remained unchangedat 9.7% (250mgpcrboar) and 12.9% (SOOmgpcrboar) duringthis period. Fvrthermore. inlheteslosteroneinjected youps. litter sizer al birth, ofgilts natually maled in August and September, were signifi-
ofabnormal
cantly higher than those of theconfrolgroup.However, a decrease in thetotalspermatozoa srcjaculalewar observed during and aner lreatment with a hi@ concentration of tcslostemne (500 mg per boar). These results suggestthat injecting suitable eonwttrations of \estostemne into boars during the hot dwnmer months wovld lx efkl~vc for the maintenance of their mpmductive perfonnancc.
INTRODLICTlON
Reduced reproductive efftciency in pigs is commonly observed during the hot summer months even in areas with a temperate climate (Okauchi and Hirakata, 1962; Thibault et al., 1966; Einarsson, 1968). Exposure ofboars to increased ambient temperatures results in a lowered courting activity (WinWeld et at., 1981; Stone, 1982) and a reduction in semen quality and fertility (McNitt and First, 1970; Wettemann et al., 1976,1979; Malmgren and La* son, 1984, Heitman et al., 1984). This decrease in semen quality during heat stress in boars is ass?ciated with reduced pregnancy rates and increased em‘Author to whom correspondence shou!d be addressed
0378-4320/91/$03.50
Q I991 -
Elsevier 5cxence Pubiishers B.V.
74
bryonic mortality (Christenson et al., i972; Wettemann et al., L976). Exposure of boars to increased ambient temperature for only 3-7 days, caused a reduction in semen quality that persisted for 6-8 weeks affer treatment ( Mazzari et al., 1968; McNitt and First, 1970; Christenson et al., 1972; Cameron and Blackshaw, 1980; Larsson and Einarsson, 1984). This phenomenon may occur in part owing to a decrease in the testosterone secretions from the testes, since the testosterone levels of boars exposed to 35°C were significantly lower than those. kept at 20°C (Larssonet al., 1983). Wettemann and Desjardins (1979) suggested that heat stress in boars can exert a transitory decline in plasma testosterone concentrations. Therefore, in this experiment, attempts were made to inhibit the reduction in semen quality by injecting testosterone into boars during the hot summer months. In addition, natural matings were performed by these boars and rcproductive performance (the conception rate and the litter sizes at birth of gilts) was also measured. MATERIALS AND METHODS
Fifteen sexually mahtre Duroc boars which had been trained for semen collection were used throughout the study. They were housed in individual pens and fed a conventional diet. None of them showed clinical signs of disease. Prior to the experiment, they were ejaculated at least once or twice weekly. These boars were divided randomly into three groups (live boars in each group). In the experimental groups, 250 or 500 mg per boar of testosterone enanthate (Enarmon Depot”, Teikoku Hormone Mgf. Co. Ltd., Tokyo) was injected into the rump muscle of boars once per month (at the end of the month) from June to September in 1988. The experimental groups will be referred to as ‘TE 250’ and ‘TE 500’ respectively. In the control group no treatment was performed.
Semen was collected by the gloved-hand technique. At collection the gel portion of the ejaculate was filtered away through double gauze and the remaining part of the ejaculate was collected in a plastic bottle. Semen was collected every 4-5 days but evaluation for semen quality was performed every 2 weeks from June to December in 1988. The volume of gel-free was measured to the nearest millilitre in a graduated plastic bottle. Sperm concentration was determined by the optical density method (Heitman et al., 1984) using a double-beam spectrophotometer (Shimadzu, Model UV-1 SO-02,Kyoto). The osmotic pressure of the semen
EFFECTSOFTESrOSTERnNEIHJcCTIONiiN
w*nsEMENQUAUTY
75
was measured using an osmometer (Orion Riken Co. Ltd., Model Osmotron5, Tokyo). Sperm motility was assessed by videomicroscopy. The suspension of spermatozoa diluted with NaCl/TES buffer ( 150 mM NaCl with 20 mM N-Tris-[hydroxymethyll-methyl-2-aminoethane sulphonic acid, pH 7.4) was placed on a microscope slide chamber, and the motility of the spermatozoa was recorded by videomicroscopy at 37°C on the thermostatically controlled warm plate. The percentages of motile spermatozoa were measured by tracing the location of the sperm heads on a transparent sheet according to the method of Katz and Overstreet (1981). The ATP concentration in spermatozoa was assayed in a boiled extract using a luminescence assay (Wishart, 1982). To examine the percentages of abnormal spermatozoa, diluted semen with NaCl/ TES buffer was spread on a microscope slide in a thin film, and the spennatozoa were fixed with formalin vapour. A total of 500 spermatozoa were examined and the number of abnormal spermatozoa (with proximal cytoplasmic droplets, abnormal sperm heads and coiled and bent tails) were counted by phase-contrast microscopy.
Using three groups of boars (control, TE 250 and TE 500), natural matings were performed with gilts (crossbred LandracexLarge White, Large WhitexLandrace), and the conception rate and the litter sizes of gilts farrowed were examined. In each group, 19-30 gilts were mated every month from June to October in 1988. Results were subjected to statistical analysis using Student’s I-test. The ambient temperature and relative humidity during the experimental periods, are shown in Fig. 1.
I.
)
Fig. Changesofaverage ambient temperature (0 and relative humidily (0 ) in aday during experimental periods. Top of the bar shows the maximum temperature and the bottom of the bar shows the minimum temperature in a day. Each point represents an average of every week.
76
H.NAIV\YAM/\ ETi\L.
RESULTS
The ejaculate volume in three groups decreased in August, although there was no significant difference among the three groups: the mean2s.e.m. (n= IS) in August of the control, TE 250 and TE 500 boars were 1072 10, 97 t 11 and 106? 14 ml, respectively (Fig. 2). The sperm motility in the three groups also decreased in the latter part of August (82.02 2.9% in the control, 77.924.846 in TE 250 and 79.0+4.4% in TE 500; the mean+s.e.m., n=S), although there was no significant difference among the three groups (Fig. 3). No distinguishable changes in the osmotic pressure of the semen were observed among the three groups throughout the experimental periods (between 295 and 314 mosmol kg-‘). Sperm concentrations decreased significantly (P~0.05) during the treatment of the TE 900 boars. They were more than 40% lower in July-October (the meanZs.e.m. during these periods was 3.34ZO.29~10~ sperm ml-‘, n=45)ascompared~thJune(5.79~0.9lx108spennml-’,n=5)forthese animals. In contrast, it was fairly constant for the control (5.65?0.05x IO*
Fig. 2. The elk1 of testosterone injection(0. control;0. 250 mg per boar;n . 500 mg peer boar) on the ejaculatevolumeof boars.Arrow showthe time of testosterone injection.Each pointrepresents an werageof fivesamplesin eachgroup.
Fig. 3. The ckt of testosterone injection(0. control:0. 2SOmg per boar;n . 5043mg per bwr) on the spwm motilityof boars.Anuws showIhe time of testosterone injection.Each pointreprcsenls an averageof fivesamplesin eachgroup.
EFFECrSOFrESTOSTERONEINJF~IONON
Bo*RSEMENQ”ALITY
77
sperm ml-’ in July-October, n=45) and TE 250 boars (5.55+0.05xIO* sperm ml-’ in July-October, n=45) (Fig. 4). There was a significant difference between the control group and the TE 500 boars (P-zO.01). Consequently, a significant decrease of total spermatozoa per ejaculate was observed during and after the treatment of the TE 500 boars (406.3 2 32.3~ lo8 spermatozoa per ejaculate in July-October, n=45) as compared with the control group (52 1.8 2 33.9~ IO’spermatozoa per ejaculate in July-October, n=45) (JkO.05). The percentage of abnormal spermatozoa in the control group increased in August to a maximum in September (34.3536%; the meants.e.m., n= 10)
Fig. 4. The elkct of testosterone injection (0. control; @. 250 mg per boar; n , 500 mg per boar) on the sperm concentrations of boars. Arrows show the time of testosterone injection. Each point represents ao average of five samples in each group.
Fig. 5. The effect of testosterone injection (0. controI: a, 250 mg per boar: 1. 500 mg per boar) on the tout percentages of abnormal spermatozoa (proximal cytoplasmic droplets plus abnormal heads and coiled and bent tails) ofboars. Arrows show the time of testosterone injection. Each poim represents an average of five samples in each group.
Fig. 6. The effecl of leslosIerone injection (0, control: @, 250 mg per boar, ., 500 mg per boar) on ATP concentrations of boar spermatozoa. Arrows show the time of te~los~emne injection. Each point represenls an average of five samples in each group.
Fig. 7. The elTec1 of tesloslemnc injection with bows (0, control: 0,250 mg per boar: l . 500 mg per boar) on the liuer sires 8t binh of fkrowing gilts (total number of piglets born) mated nawrally in each month. Each poinl representsannverageof I&28giltsfarrowed in each group. *‘PcO.CI.
lPcO.O5
compared
with the control.
declined thereafter (Fig. 5). In contrast, the percentages of abnormal spermatozoa in the testosterone-injected groups remained fairly low during this period (9.7 + 0.9% in TE 250 and 12.9? 1.3%in TE 500), and there were signitlcant differences between the control group and the testosterone-injected group (P-=0.01 ). ATP concentrations ofspermatozoa in the control group decreased in July reaching their lowest level in August: the meanZs.e.m. (n= IS) in August was 8924.3 nmol per IO9sperm. However, no remarkable deeline was observed in theTE 250 (I 1659.8 nmolper IO9sperm) andTE 500 (I 1428.7 nmol per 10’)sperm) boars in August (Fig. 6). There were significant differences between the control group and the testosterone-injected goups (P
gust, n=22; 10.3ZO.5 in September, n=22) and TE 500 (11.320.7in August, n=26; 11.7?0.8 in September, n=22) boars, although these had returned to levels similar to the testosterone-injected groups in October. The conception rates for the control boars were 73 113% (the mean 5s.e.m. of the percentage of gilts pregnant for each boar, n = 5) in August and 87 + 3% in September, whereas values of 85 2 6% (TE 250) and 9 1i7% (TE 500) in August, and 9228% (TE 250) and 8827% (TE 500) in September were obtained in the testosterone-injected groups. However, there was no significant difference among the three groups in each month. DISCUSSION
It has been observed that high ambient temperature is associated with reduced semen quality and fertility in boars (Hughes and Yarley, 1980). It is a serious problem that the litter sizes of sows mated during and after the hot summer months decrease significantly in many of the pig-producing areas of sub-tropical and tropical regions in Japan. However, the present study suggested that testosterone injection in boars during the hot summer months would be useful for the maintenance of semen quality, especially the incidence of abnormal spermatozoa, and the reproductive performance of boars. Exposure of boars to an increased ambient temperature has been reported to cause decreased ejaculate volume, total sperm output and percentage of motile spermatozoa (Okauchi and Hirakata, 1962; Christenson et al., 1972; Stone, 1982), but in other experiments, ejaculate volume and total sperm output have remained unaffected (Wettemann et al., 1976; Camerou and Blackshaw, 1980; Larsson and Einarsson, 1984). However, an increase of abnormal spermatozoa has been found in all reports (e.g. Christenson et al., 1972; Wettemann et al., 1976; Cameron and Blackshaw, 1980; Egbunike and Dede, :9&3; Larsson and Einarsson, 1984; Heitman et al., 1984; Malmgren and Larsson, 1984). The present results also showed that the most remarkable changes in semen quality during and aRer the hot summer months were obtained in the incidence of abnormal spermatozoa. Spermatozoa in the control group showed proximal cytoplasmic droplets, abnormal heads and coiled and bent tails. In contrast, it was found that testosterone injection in boars during the hot summer months inhibited the increased production of abnormal spermatozoa. In addition to the incidence of abnormal spermatozoa, it is interesting to note that ATP concentrations of the spermatozoa in the control group decreased significantly in August compared with those of the testosterone-injected groups (Fig. 6). The reasons have however remained unsolved. Further study is therefore needed to examine the correlations between ATP concentrations and the fertilizing ability of boar spermatozoa. Comhaire et di. ( 1983) proposed that the measurement of semen ATP may be a possible
80
H.
NAKAYAMA
ETAL
biochemical method for the quantification of the actual fertilizing potential of human ejaculate. The present study has not measured the changes of testosterone levels in boars during the experimental periods. However, it has been reported that the testosterone levels of boars exposed to 35°C were significantly lower than those kept at 20°C (Lamson et al., 1983). Androgen biosynthesis by slices of testis tissue from boars exposed to elevated ambient temperature for 90 days was significantly lower compared with control boars ( Wettemann and Desjardins, 1979). These results suggest that elevated ambient temperatures alter testicular endocrine activity. A relatively high level of testosterone is required for the maintenance of sperm maturation in the epididymis (Garner and Hafez, 1987). Furthermore, the high incidence of proximal cytoplasmic droplets may be caused by the long-term effect of elevated ambient temperature on the epididymis which, because of its unique location in the scrotum in the boars, is most sensitive to temperature (Jainudeen and Hafez, 1987). In this study, the replenishment of endogenous testosterone levels by injecting exogenous testosterone enanthate might inhibit an increased incidence of proximal cytoplasmic droplets, abnormal heads and, coiled and bent tails. However, in addition to the positive effects of testosterone injection, a negative one has been presented. The oroduction of spermatozoa calculated from sperm concentration and ejaculate volume (Figs. 2 and 4) decreased during and after treatment with the high concentration of testosterone (TE 500). It might be a symptom of the negative feedback control of testosterone on gonadotrophin secretion. In the present work, enough spermatozoa for fertilization would be ejaculated per one mating, since natural matings were performed. Therefore, the concep tion rates did not decline in the TE 500 boars. Conversely, litter sizes at birth of gilts were significantly higher than those of the control group in August and September. However, if artificial insemination is used for breeding, the production of spermatozoa might be of economical importance when considering the possible number of semen doses which can be prepared from one boar. Therefore, further study is needed to determine the most suitable concentrations and frequency for testosterone injection. ACKNOWLEDGEMENTS
We thank Dr. G.J. Wishart, Dundee Institute ofTechnology, Scotland, U.K., for rendinn the munuscriot. The testosterone enanthate used in this research was don&d by Mr. I. M&takata, Teikoku Hormone Mgf. Co. Ltd., Fukuoka, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ito Memorial Foundation.
EFFECTSOF TESTOSTERONE lNlECTIOV
ON SOAR SEMEN QVLlTV
81
REFERENCES Cameron, R.D.A. and Blackshaw, A.W.. 1980. The effect of elevated ambient temperature on spwntatogenesisin theboar. J. Repmd. Fenil.. 59: 173-179. Christenson. R.K., Teague. H.S.. Grifo. A.P.. Jr. and Roller. W.L., 1972. The effect of high environmental temperature on the boar. Ohio Agric. Res. Dev. Center. Wwxter. OH. Ohio Swine Research and Information Report: Research Summary 61, pp. 19-23. Comhaw F.. Venneule”. L.. Ghedira, K., Mas, J.. Irvine. S. and Callipolitis, G.. 1953. Admosine triphosphate in human semen: a quantitative estimate of fertilizing potential. Fertil. steri1.. 4c: 500-504. Egbunike. G.N. and Dede, T.L. !980. The intluence ofshon-term exposure to tmpical sunlight on hoar seminal characteristics. Int. I. Biometeoral.. 24: 129-135. Einarsson, S., 1968. Factors affecting fertility in artificial insemination of swine. Nerd. Veteti“aemted.. 20: 622-629. Garner, D.L. and Hafez. E.S.E.. 1987. Spemxatozoa and seminal plasma. In: E.S.E. Hafez (Edilor). Reproduction in Farm Animals, Lea and Febiger. Philadelphia, PA, Sth edn., pp. I89209. Heitman, H, Jr., Cockrell. J.R. and Morrison, S.R., 1984. Cycling ambient temperature effect on boar semen. Anim. Prod., 38: 129-l 32. Hughes. P.E. and Varley, M.A. (Editors ), 1980. Reproduction in the Pig. Butteworth, London. pp. 173-19s. Jainudee”. MR. and Hafez. E.S.E.. 1987. Reproductive failure in males. In: E.S.E. Hafez (Editor), Reproduction in Farm Animals, Lea and Febiger. Philadelphia, PA, 5th edn., pp. 423435.
Sperm
Katz. D.F. and Overstreet, J.W., 1981. motility assessment by videomicmgraphy. Fertil. Sterii.. 35: 188-193. lxsson. K. and Einarsson, S., 1984. Seminal changes in boars after heat stress. Acta Vet. Stand., 25: 57-66. Larsson. K.. Einarsson. S.. Lundstrom. K. and Hakkarainen. 1.. 1983. Endocrine etTectsof heat stress in boars. Acta Vet. Stand.. 24: 305-3 14. Malmgren, L. and Larsson, K., 1984. Semen quality and fertility after heat stress in boars. Acla Vet. Stand., 25: 425-435. Mazrari. G.. du Mesnil du Buisson, F. and Onavant. R.. 1968. Action of temperature and light on spcrmatogenesir. sperm production and fertility of the boar. Proc. 6th Int. Congr. Anim. Repmd. and AI. Paris. vol. I, pp. 305-308. McNitt. J.I. and First. N.L., 1970. Effects of 72.hour heat stress on semen quality in boars. Int. J. Biometeoral., 14: 373-360. Okauchi, K. and Himkata, K.. 1962. Studies on the characters of boar semen. 1. Effects of environmental temperature. Bu!!. Fat. Agric.. Miyazaki Univ., 7: 117-123. Stone, B.A., 1982. Heat induced infertility of baan: the inter-relationship between depressed sperm output and fertility and a” estimation of the critical temperature above which sperm output is impaired. Anim. Reprod. Sci.. 4: 283-299. Thibault. C.M. Courot. M., Maninet. L., Maulwn, P.. du Mesnil du Buisson, F., Onavant, R.. Pelleticr. J. and Signoret, J.P., 1966. Regtdaticn of breeding season ar.d estroos cycles by light and external stimuli in some mammals. J. Anim. Sci., 25: Suppl. 119-142. Wettemann. R.P. and Desjardin: C., 1979. Testicular function in boars exposed to elevated ambient temperature. Biol. Repmd., 20: 235-241. Wettemann. R.P., Wella M.E., Omtvedt, LT., Pope, C.E. and Tutman, E.J., 1976. Influenceof elevated ambient temperatureon reproductiveperfomlsnce ofboars. J. Anin. Sci., 42: 664669. Wettemann, R.P.. Wells, ME. and Johnson, R.K., 1979. Reproductive character&b of boars during and after exposure to increased ambient temperature. J. Anim. ki., 49: 1501-l 505.
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Winfield, C.G., Hemsworth, P.H.. Galloway, D.B. and Makin. A.W.. 1981. Sexual behaviour and semen characteristics of boars: effects of high temperature. Aust. J. Exp. Agtic. Anim. Hush., 21: 39-45. Wishan, G.J., 1982. MaintenanceofATPconccnrrations inand offeniiizing ability of fowl and tarkey spermarozoa in vitro. J. Reprcd. Fertil.. 66: 457-462.
73
Effects of testosterone injection on the semen quality in boars during high ambient temperature
ABSTRACT NPkayama. Ii.. Hidaka. R. and Ashizawa. K.. 1991. Effects of tatosterone injection on the semen quality in boars duting high ambient temperature. Anin?, Rept(h;. Sci, 25: 73-82. Attempts were made to inhibit the reduction in wnen quality and reproductive pcrfoormanceby injecting testoslemne into boars during the hot summer months. Testosterone enanthate (250 cn 500 mg pr boar) was injccled into the rump muscle ofboan once a month from June to September. The most t’cmarirabIecffectr were obtained in rhe incidwtce ofabnormat spermatows. In themnlml group (no injnian). the percentagegel spermatozoa i.e. those with proximal cytoplasmif drop lets. abnommt heads and roiled and bent tails increased to 34.3 f 3.6% in September. In ~ntrasl, the pmntaacs of abnormal spermatozoa in the testostemne-injectedamups remained unchangedat 9.7% (250mgpcrboar) and 12.9% (SOOmgpcrboar) duringthis period. Fvrthermore. inlheteslosteroneinjected youps. litter sizer al birth, ofgilts natually maled in August and September, were signifi-
ofabnormal
cantly higher than those of theconfrolgroup.However, a decrease in thetotalspermatozoa srcjaculalewar observed during and aner lreatment with a hi@ concentration of tcslostemne (500 mg per boar). These results suggestthat injecting suitable eonwttrations of \estostemne into boars during the hot dwnmer months wovld lx efkl~vc for the maintenance of their mpmductive perfonnancc.
INTRODLICTlON
Reduced reproductive efftciency in pigs is commonly observed during the hot summer months even in areas with a temperate climate (Okauchi and Hirakata, 1962; Thibault et al., 1966; Einarsson, 1968). Exposure ofboars to increased ambient temperatures results in a lowered courting activity (WinWeld et at., 1981; Stone, 1982) and a reduction in semen quality and fertility (McNitt and First, 1970; Wettemann et al., 1976,1979; Malmgren and La* son, 1984, Heitman et al., 1984). This decrease in semen quality during heat stress in boars is ass?ciated with reduced pregnancy rates and increased em‘Author to whom correspondence shou!d be addressed
0378-4320/91/$03.50
Q I991 -
Elsevier 5cxence Pubiishers B.V.
74
bryonic mortality (Christenson et al., i972; Wettemann et al., L976). Exposure of boars to increased ambient temperature for only 3-7 days, caused a reduction in semen quality that persisted for 6-8 weeks affer treatment ( Mazzari et al., 1968; McNitt and First, 1970; Christenson et al., 1972; Cameron and Blackshaw, 1980; Larsson and Einarsson, 1984). This phenomenon may occur in part owing to a decrease in the testosterone secretions from the testes, since the testosterone levels of boars exposed to 35°C were significantly lower than those. kept at 20°C (Larssonet al., 1983). Wettemann and Desjardins (1979) suggested that heat stress in boars can exert a transitory decline in plasma testosterone concentrations. Therefore, in this experiment, attempts were made to inhibit the reduction in semen quality by injecting testosterone into boars during the hot summer months. In addition, natural matings were performed by these boars and rcproductive performance (the conception rate and the litter sizes at birth of gilts) was also measured. MATERIALS AND METHODS
Fifteen sexually mahtre Duroc boars which had been trained for semen collection were used throughout the study. They were housed in individual pens and fed a conventional diet. None of them showed clinical signs of disease. Prior to the experiment, they were ejaculated at least once or twice weekly. These boars were divided randomly into three groups (live boars in each group). In the experimental groups, 250 or 500 mg per boar of testosterone enanthate (Enarmon Depot”, Teikoku Hormone Mgf. Co. Ltd., Tokyo) was injected into the rump muscle of boars once per month (at the end of the month) from June to September in 1988. The experimental groups will be referred to as ‘TE 250’ and ‘TE 500’ respectively. In the control group no treatment was performed.
Semen was collected by the gloved-hand technique. At collection the gel portion of the ejaculate was filtered away through double gauze and the remaining part of the ejaculate was collected in a plastic bottle. Semen was collected every 4-5 days but evaluation for semen quality was performed every 2 weeks from June to December in 1988. The volume of gel-free was measured to the nearest millilitre in a graduated plastic bottle. Sperm concentration was determined by the optical density method (Heitman et al., 1984) using a double-beam spectrophotometer (Shimadzu, Model UV-1 SO-02,Kyoto). The osmotic pressure of the semen
EFFECTSOFTESrOSTERnNEIHJcCTIONiiN
w*nsEMENQUAUTY
75
was measured using an osmometer (Orion Riken Co. Ltd., Model Osmotron5, Tokyo). Sperm motility was assessed by videomicroscopy. The suspension of spermatozoa diluted with NaCl/TES buffer ( 150 mM NaCl with 20 mM N-Tris-[hydroxymethyll-methyl-2-aminoethane sulphonic acid, pH 7.4) was placed on a microscope slide chamber, and the motility of the spermatozoa was recorded by videomicroscopy at 37°C on the thermostatically controlled warm plate. The percentages of motile spermatozoa were measured by tracing the location of the sperm heads on a transparent sheet according to the method of Katz and Overstreet (1981). The ATP concentration in spermatozoa was assayed in a boiled extract using a luminescence assay (Wishart, 1982). To examine the percentages of abnormal spermatozoa, diluted semen with NaCl/ TES buffer was spread on a microscope slide in a thin film, and the spennatozoa were fixed with formalin vapour. A total of 500 spermatozoa were examined and the number of abnormal spermatozoa (with proximal cytoplasmic droplets, abnormal sperm heads and coiled and bent tails) were counted by phase-contrast microscopy.
Using three groups of boars (control, TE 250 and TE 500), natural matings were performed with gilts (crossbred LandracexLarge White, Large WhitexLandrace), and the conception rate and the litter sizes of gilts farrowed were examined. In each group, 19-30 gilts were mated every month from June to October in 1988. Results were subjected to statistical analysis using Student’s I-test. The ambient temperature and relative humidity during the experimental periods, are shown in Fig. 1.
I.
)
Fig. Changesofaverage ambient temperature (0 and relative humidily (0 ) in aday during experimental periods. Top of the bar shows the maximum temperature and the bottom of the bar shows the minimum temperature in a day. Each point represents an average of every week.
76
H.NAIV\YAM/\ ETi\L.
RESULTS
The ejaculate volume in three groups decreased in August, although there was no significant difference among the three groups: the mean2s.e.m. (n= IS) in August of the control, TE 250 and TE 500 boars were 1072 10, 97 t 11 and 106? 14 ml, respectively (Fig. 2). The sperm motility in the three groups also decreased in the latter part of August (82.02 2.9% in the control, 77.924.846 in TE 250 and 79.0+4.4% in TE 500; the mean+s.e.m., n=S), although there was no significant difference among the three groups (Fig. 3). No distinguishable changes in the osmotic pressure of the semen were observed among the three groups throughout the experimental periods (between 295 and 314 mosmol kg-‘). Sperm concentrations decreased significantly (P~0.05) during the treatment of the TE 900 boars. They were more than 40% lower in July-October (the meanZs.e.m. during these periods was 3.34ZO.29~10~ sperm ml-‘, n=45)ascompared~thJune(5.79~0.9lx108spennml-’,n=5)forthese animals. In contrast, it was fairly constant for the control (5.65?0.05x IO*
Fig. 2. The elk1 of testosterone injection(0. control;0. 250 mg per boar;n . 500 mg peer boar) on the ejaculatevolumeof boars.Arrow showthe time of testosterone injection.Each pointrepresents an werageof fivesamplesin eachgroup.
Fig. 3. The ckt of testosterone injection(0. control:0. 2SOmg per boar;n . 5043mg per bwr) on the spwm motilityof boars.Anuws showIhe time of testosterone injection.Each pointreprcsenls an averageof fivesamplesin eachgroup.
EFFECrSOFrESTOSTERONEINJF~IONON
Bo*RSEMENQ”ALITY
77
sperm ml-’ in July-October, n=45) and TE 250 boars (5.55+0.05xIO* sperm ml-’ in July-October, n=45) (Fig. 4). There was a significant difference between the control group and the TE 500 boars (P-zO.01). Consequently, a significant decrease of total spermatozoa per ejaculate was observed during and after the treatment of the TE 500 boars (406.3 2 32.3~ lo8 spermatozoa per ejaculate in July-October, n=45) as compared with the control group (52 1.8 2 33.9~ IO’spermatozoa per ejaculate in July-October, n=45) (JkO.05). The percentage of abnormal spermatozoa in the control group increased in August to a maximum in September (34.3536%; the meants.e.m., n= 10)
Fig. 4. The elkct of testosterone injection (0. control; @. 250 mg per boar; n , 500 mg per boar) on the sperm concentrations of boars. Arrows show the time of testosterone injection. Each point represents ao average of five samples in each group.
Fig. 5. The effect of testosterone injection (0. controI: a, 250 mg per boar: 1. 500 mg per boar) on the tout percentages of abnormal spermatozoa (proximal cytoplasmic droplets plus abnormal heads and coiled and bent tails) ofboars. Arrows show the time of testosterone injection. Each poim represents an average of five samples in each group.
Fig. 6. The effecl of leslosIerone injection (0, control: @, 250 mg per boar, ., 500 mg per boar) on ATP concentrations of boar spermatozoa. Arrows show the time of te~los~emne injection. Each point represenls an average of five samples in each group.
Fig. 7. The elTec1 of tesloslemnc injection with bows (0, control: 0,250 mg per boar: l . 500 mg per boar) on the liuer sires 8t binh of fkrowing gilts (total number of piglets born) mated nawrally in each month. Each poinl representsannverageof I&28giltsfarrowed in each group. *‘PcO.CI.
lPcO.O5
compared
with the control.
declined thereafter (Fig. 5). In contrast, the percentages of abnormal spermatozoa in the testosterone-injected groups remained fairly low during this period (9.7 + 0.9% in TE 250 and 12.9? 1.3%in TE 500), and there were signitlcant differences between the control group and the testosterone-injected group (P-=0.01 ). ATP concentrations ofspermatozoa in the control group decreased in July reaching their lowest level in August: the meanZs.e.m. (n= IS) in August was 8924.3 nmol per IO9sperm. However, no remarkable deeline was observed in theTE 250 (I 1659.8 nmolper IO9sperm) andTE 500 (I 1428.7 nmol per 10’)sperm) boars in August (Fig. 6). There were significant differences between the control group and the testosterone-injected goups (P
gust, n=22; 10.3ZO.5 in September, n=22) and TE 500 (11.320.7in August, n=26; 11.7?0.8 in September, n=22) boars, although these had returned to levels similar to the testosterone-injected groups in October. The conception rates for the control boars were 73 113% (the mean 5s.e.m. of the percentage of gilts pregnant for each boar, n = 5) in August and 87 + 3% in September, whereas values of 85 2 6% (TE 250) and 9 1i7% (TE 500) in August, and 9228% (TE 250) and 8827% (TE 500) in September were obtained in the testosterone-injected groups. However, there was no significant difference among the three groups in each month. DISCUSSION
It has been observed that high ambient temperature is associated with reduced semen quality and fertility in boars (Hughes and Yarley, 1980). It is a serious problem that the litter sizes of sows mated during and after the hot summer months decrease significantly in many of the pig-producing areas of sub-tropical and tropical regions in Japan. However, the present study suggested that testosterone injection in boars during the hot summer months would be useful for the maintenance of semen quality, especially the incidence of abnormal spermatozoa, and the reproductive performance of boars. Exposure of boars to an increased ambient temperature has been reported to cause decreased ejaculate volume, total sperm output and percentage of motile spermatozoa (Okauchi and Hirakata, 1962; Christenson et al., 1972; Stone, 1982), but in other experiments, ejaculate volume and total sperm output have remained unaffected (Wettemann et al., 1976; Camerou and Blackshaw, 1980; Larsson and Einarsson, 1984). However, an increase of abnormal spermatozoa has been found in all reports (e.g. Christenson et al., 1972; Wettemann et al., 1976; Cameron and Blackshaw, 1980; Egbunike and Dede, :9&3; Larsson and Einarsson, 1984; Heitman et al., 1984; Malmgren and Larsson, 1984). The present results also showed that the most remarkable changes in semen quality during and aRer the hot summer months were obtained in the incidence of abnormal spermatozoa. Spermatozoa in the control group showed proximal cytoplasmic droplets, abnormal heads and coiled and bent tails. In contrast, it was found that testosterone injection in boars during the hot summer months inhibited the increased production of abnormal spermatozoa. In addition to the incidence of abnormal spermatozoa, it is interesting to note that ATP concentrations of the spermatozoa in the control group decreased significantly in August compared with those of the testosterone-injected groups (Fig. 6). The reasons have however remained unsolved. Further study is therefore needed to examine the correlations between ATP concentrations and the fertilizing ability of boar spermatozoa. Comhaire et di. ( 1983) proposed that the measurement of semen ATP may be a possible
80
H.
NAKAYAMA
ETAL
biochemical method for the quantification of the actual fertilizing potential of human ejaculate. The present study has not measured the changes of testosterone levels in boars during the experimental periods. However, it has been reported that the testosterone levels of boars exposed to 35°C were significantly lower than those kept at 20°C (Lamson et al., 1983). Androgen biosynthesis by slices of testis tissue from boars exposed to elevated ambient temperature for 90 days was significantly lower compared with control boars ( Wettemann and Desjardins, 1979). These results suggest that elevated ambient temperatures alter testicular endocrine activity. A relatively high level of testosterone is required for the maintenance of sperm maturation in the epididymis (Garner and Hafez, 1987). Furthermore, the high incidence of proximal cytoplasmic droplets may be caused by the long-term effect of elevated ambient temperature on the epididymis which, because of its unique location in the scrotum in the boars, is most sensitive to temperature (Jainudeen and Hafez, 1987). In this study, the replenishment of endogenous testosterone levels by injecting exogenous testosterone enanthate might inhibit an increased incidence of proximal cytoplasmic droplets, abnormal heads and, coiled and bent tails. However, in addition to the positive effects of testosterone injection, a negative one has been presented. The oroduction of spermatozoa calculated from sperm concentration and ejaculate volume (Figs. 2 and 4) decreased during and after treatment with the high concentration of testosterone (TE 500). It might be a symptom of the negative feedback control of testosterone on gonadotrophin secretion. In the present work, enough spermatozoa for fertilization would be ejaculated per one mating, since natural matings were performed. Therefore, the concep tion rates did not decline in the TE 500 boars. Conversely, litter sizes at birth of gilts were significantly higher than those of the control group in August and September. However, if artificial insemination is used for breeding, the production of spermatozoa might be of economical importance when considering the possible number of semen doses which can be prepared from one boar. Therefore, further study is needed to determine the most suitable concentrations and frequency for testosterone injection. ACKNOWLEDGEMENTS
We thank Dr. G.J. Wishart, Dundee Institute ofTechnology, Scotland, U.K., for rendinn the munuscriot. The testosterone enanthate used in this research was don&d by Mr. I. M&takata, Teikoku Hormone Mgf. Co. Ltd., Fukuoka, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ito Memorial Foundation.
EFFECTSOF TESTOSTERONE lNlECTIOV
ON SOAR SEMEN QVLlTV
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