Seasonal variation in semen characteristics, scrotal circumference and libido of Persian Karakul rams

Small Ruminant Research 53 (2004) 133–139

Technical note

Seasonal variation in semen characteristics, scrotal circumference and libido of Persian Karakul rams M. Kafi a,∗ , M. Safdarian b , M. Hashemi b a

Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz 71345, Iran b Fars Research Center for Agricultural Natural Resources and Animal Husbandry, 71555, Iran Received 11 October 2002; received in revised form 17 June 2003; accepted 18 July 2003

Abstract The aim of the present study was to evaluate seasonal changes in semen characteristics of Persian Karakul rams. Six Karakul rams (3–4 years old) were trained to serve the artificial vagina. Semen collection was performed every 2 weeks, commencing in January (at onset of winter) to December (end of autumn) 1999. Semen ejaculates were evaluated for volume, total sperm output, mass motility and the percentage live sperm. Moreover, changes in the scrotal circumference, body weights, libido and plasma testosterone concentrations of the rams were also recorded at 2-week intervals. The semen was characterized by a mean (±S.D.) ejaculate volume of 1.2 ± 0.3 ml, a mean (±S.D.) mass motility of 3.8 ± 0.8, and a total number of 4442 ± 1247 (×106 ) sperm per ejaculate for the observation period. The overall mean (±S.D.) scrotal circumference recorded was 32.0 ± 1.2 cm. Seasonal variations (P < 0.05) were recorded for semen volume, mass motility, scrotal circumferences and plasma testosterone concentrations, but not for the total sperm output. In conclusion, albeit there were seasonal changes in semen characteristics of Karakul rams, the semen has the capability and quality to be used for artificial insemination all year round. Semen of superior quality and quantity was especially collected in late summer and throughout autumn. © 2003 Elsevier B.V. All rights reserved. Keywords: Semen; Scrotal circumference; Karakul rams; Libido

1. Introduction It has been observed that all breeds of male sheep living above 40◦ latitude in the northern hemisphere display seasonal variation in testicular volume and daily sperm production (Pelletier et al., 1988; Ringwall et al., 1990). The reproductive activity of the ram appears to be influenced, in certain breeds and regions, by the season of the year (Boland et al., 1985; Glover et al., 1990; Karagiannidis et al., 2000) with photoperiod being the key environmental signal tim∗ Corresponding author. Tel.: +98-711-6280704; fax: +98-711-6280707. E-mail address: [email protected] (M. Kafi).

ing the reproductive cycle (Lincoln and Short, 1980). It is also important to stress that under certain rearing conditions (i.e. extensive management), level of nutrition may have a greater influence than photoperiod (Mukasa-Mugerwa and Ezaz, 1992; Perez et al., 1997). Karakul sheep are one of the most economically important skin and meat-breed of sheep reared in Iran. Little is known regarding the reproductive performance of Karakul rams and ewes under normal environmental conditions in Iran. Due to the desire to use AI in sheep production systems in southern Iran during various seasons, the current study was designed to determine seasonal variations in semen characteristics of the Karakul rams. In addition

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data were collected on testosterone profile, scrotal measurements (SC) and libido associated with season of the year.

corresponding live weight (Gastel et al., 1995). SC was also measured using a flexible tape at the widest scrotal diameter at 2-week intervals. 2.3. Semen collection and evaluation

2. Materials and methods 2.1. Location and climate The study was carried out from January to December 1999, on a farm located in Ab-Barik district in north of Shiraz, Fars province at 29◦ 50 N, 52◦ 46 E and an altitude of 1630 m. Climatological information regarding this location during the course of the study, is summarized in Table 1. 2.2. Animals Six rams of the Karakul breed, 3–4 years of age and an average live weight of 75.0±6.3 kg were randomly selected from the breeding flock. All animals were kept indoors in a covered shelter and allowed to walk freely. A general management program including deworming, disease prevention and hoof trimming was applied. Animals were clinically examined regarding the health of their external genitalia. Rams were fed a mixture of straw–hay and barley according to the recommended requirements of the NRC throughout the year. Daily amounts provided per head were 0.8 kg hay, 1120 g alfa alfa and 900 g barley—these were given in two equal quantities per day. Water and sheep mineral blocks were available ad libitum. Rams were weighed at fortnightly intervals. The animals were not used for any breeding program during the course of the study. Monthly fleece growth were estimated and then subtracted from each body weight to obtain the Table 1 Mean (±S.D.) climatological data for the experimental year from the Shiraz Meteorological Center Season

Winter Spring Summer Autumn a

Air Relative temperature humidity (◦ C) (%) 5.8 17.3 26.0 14.3

± ± ± ±

3.8 4.1 4.8 5.3

66.4 47.2 28.8 40.0

± ± ± ±

6.2 11.5 2.6 16.2

Temperature–humidity index.

Day length

THIa

The animals were trained to serve the artificial vagina. The training period lasted 6–8 weeks. An ovariectomized ewe was used as a teaser and rams were placed in a pen adjacent to the collection area where the semen collection procedure could be visualized by each ram (Evans and Maxwell, 1987). Each ram was scheduled on alternate weeks on the same day for semen collection. Semen samples were evaluated for volume (ml), mass motility (on a scale of 0 (immotile) to 5 (vigorous motility)), density, total sperm output and the percentage live sperm cells. Semen smears were immediately prepared, and another aliquot of the semen sample was diluted with buffered saline solution (Evans and Maxwell, 1987). The density was measured with an EEL colorimeter calibrated against haemocytometer counts. Total number of sperm per ejaculate was calculated by multiplying sperm density and ejaculate volume. The proportion of live and dead spermatozoa was determined using the nigrosin-eosin staining technique by counting at least 200 spermatozoa under an oil immersion objective (1000×) random fields. All examinations were performed by the same operator. 2.4. Libido Every 2 weeks the rams were evaluated for the degree of libido, using three ovariectomized ewes. Ewes were pretreated with 50 mg progesterone acetate (Abouraihan Co; Iran) for 8 days (every other day) and 2 mg estradiol benzoate (Abouraihan Co; Iran) on the last day of progesterone therapy. Each ram was exposed to three ovariectomized ewes for a 20-min period and then the number of mounts and successful matings were recorded (Holmes, 1986). 2.5. Blood sampling and hormone assay

10.9 13.2 14.6 11.3

± ± ± ±

0.6 1.2 0.7 0.6

10.0 23.4 32.4 18.5

± ± ± ±

2.3 4.2 2.1 5.6

A jugular blood sample was collected from each ram in heparinized tubes and immediately placed on ice. The blood samples were transported to the laboratory and centrifuged at 3500 rpm for 10 min at 4 ◦ C.

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The harvested plasma was then stored at −20 ◦ C until analyzed for plasma testosterone concentrations. The concentration of testosterone in the plasma was measured in all samples by enzyme immunoassay using a diagnostic commercial kit (Testosterone Enzyme Immunoassay Test Kit, Medix Biotech Inc., CA). The reagents had been previously validated for sheep plasma using a parallelism test. The detection limits of the assay was 0.2–16.0 ng/ml. The intraand inter-assay coefficients of variations were 6.5 and 9.3%, respectively.

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measures analysis of variance was performed to statistically evaluate any marked changes in dependant variables (Everitt, 1995). Duncan’s method was used to compare means (Steel and Torrie, 1980). Pearson correlation coefficients among the seminal parameters were also investigated. The level of significance was set at P < 0.05 for all tests.

3. Results Changes in SC of the rams are set out in Table 2. Mean SC varied significantly (P < 0.05) throughout the year, with the lowest mean value during winter (31.1 ± 1.6 cm) and the highest value being recorded during autumn (33.3 ± 1.4 cm). The mean SC significantly increased (P < 0.05) from the end of winter to spring. Another significant increase in SC (P < 0.05) was observed at the end of spring towards early autumn (June–October) (Fig. 1A). Changes in the live weight during the study were not significant (Fig. 1B). Furthermore, no significant correlation between live weight and SC in the rams was recorded. A total number of 156 semen ejaculates were collected from the six Karakul rams during this study. Semen volume was significantly affected by month and season of the year (P < 0.01). Mean semen volume increased significantly (P < 0.05) from the end of summer and remained high during autumn. The highest value of the mean semen volume (1.6±0.5 ml) was

2.6. Statistical analysis Data were expressed as means (±S.D.) and analyzed using software of statistical analysis system (SAS, 1990) according to the following liner model: Yijkl = N + Si + Mj (Si ) + Rk (RS)ik +Rk Mj (Si ) + Eijkl where Yijkl is the dependent variable, N the general mean, Si the effect of season, Mj (S)i the effect of the jth month (within season), Rk the effect of kth ram, (RS)ik the ram × season interaction, Rk Mj (Si ) the ram × month (within season) interaction and Eijkl the random error. Dependent variables in the mathematical model were semen volume, total sperm output, mass motility, the percentage of live sperm, plasma testosterone concentrations and SC. Summary Table 2 Mean (±S.D.) seminal characteristics in Karakul ramsa Season

Semen volume (ml)

Winter Spring Summer Autumn

1.3 1.0 1.1 1.3

± ± ± ±

0.2 0.2 0.2 0.3

a b b a

3.6 3.6 3.7 4.2

± ± ± ±

0.7 0.9 0.8 0.8

b b b a

87.4 90.8 93.5 91.7

± ± ± ±

0.9 a 0.9 0.6 b 0.9

4778 4262 4161 4570

± ± ± ±

1260 1050 1020 1660

31.1 31.9 32.5 33.3

± ± ± ±

1.6 b 1.2 1.0 1.4 a

Ram 1 2 3 4 5 6 Overall mean ± S.D.

1.2 1.1 1.3 1.2 1.1 1.3 1.2

± ± ± ± ± ± ±

0.4 0.5 0.2 0.2 0.1 a 0.5 b 0.3

3.4 4.0 4.0 4.0 4.2 3.5 3.8

± ± ± ± ± ± ±

0.8 0.9 0.6 0.9 1.0 0.7 0.8

a b b b b a

91.6 90.4 89.7 89.7 91.2 88.5 90.2

± ± ± ± ± ± ±

2.9 3.0 2.8 3.1 2.8 3.0 2.9

4607 4416 4905 3995 3931 4774 4442

± ± ± ± ± ± ±

1102 ab 1205 ab 850 a 967 b 675 b 1232 ab 1247

31.8 30.9 31.4 33.2 32.8 33.0 32.0

± ± ± ± ± ± ±

0.8 1.3 1.4 0.8 1.9 1.0 1.2

a

Mass activity

Live sperm cell (%)

Total sperm output (×106 )

Scrotal circumference (cm)

Means for each effect in the same column, with different superscript differ significantly (P < 0.05).

a a a b a b

Testosterone (ng/ml) 3.6 4.1 10.3 6.2

± ± ± ±

0.8 1.3 2.2 1.2

a a b a

5.6 6.2 6.3 7.7 4.2 5.7 6.0

± ± ± ± ± ± ±

0.8 0.8 0.4 1.0 0.6 0.6 0.7

a a a a b a

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Fig. 1. Monthly changes in scrotal circumference (A), live weight (B) and testosterone concentrations (C) in six Karakul rams (January–December 1999) (mean ± S.D.).

recorded in December. A significant decrease (P < 0.01) in the mean semen volume was observed from the middle of spring towards the end of summer. The lowest volume of semen was that in May (0.9±0.1 ml). The correlation coefficient between semen volume and total sperm output was highly significant (r = 0.85; P < 0.01). Changes in the semen volume could ex-

plain more than 70% (r 2 = 0.72) of the variation in the total sperm output. Month and season had a significant effect on mass motility (P < 0.01). Semen samples in autumn had a significantly higher mass motility (P < 0.05) compared to values obtained in the other seasons. The lowest value for mass motility (3.3 ± 0.8) was

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recorded in August. Mean mass motility was significantly correlated with the total sperm output and percent live sperm (r = 0.24 and r = −0.48; P < 0.01), respectively. The variation between rams in scrotal circumference and mass motility was significant (P < 0.05) (Table 2). A significant season–ram interaction (P < 0.05) was observed in scrotal circumference. Similarly, the month (within season)–ram interaction was significant (P < 0.05) for scrotal circumference. The total sperm output was not affected by month. The lowest and the highest percentage of live sperm were recorded in September (97.6 ± 13.1) and in January (82.1 ± 11.3), respectively. The semen samples with a higher total sperm output had a tendency for a higher mass motility. The concentration of plasma testosterone was influenced by month and season of the year (P < 0.01) but not by the ram. Plasma testosterone concentration declined (P < 0.05) in December and remained low until May with the lowest mean concentration (4.0 ± 0.7 ng/ml) recorded in January. A significant increase was observed in the mean concentration of testosterone from May to August (P < 0.05). This increase was observed 1 month before the occurrence of a significant increase in mean SC of the rams (Fig. 1A and C). Season and month had no significant influence on the number of mounts however, the number of matings of the rams was significantly (P < 0.05) lower in autumn (2.5±0.6) than in winter (7.0±1.6). Summer temperature had no significant effect on the number of matings.

4. Discussion The effect of season and/or day length on semen quality has been studied in different breeds of rams (Colas, 1980; Boland et al., 1985; Amir et al., 1986; Ibrahim, 1997). This study is the first to report the seasonal changes in reproductive indices of Karakul rams reared in southern Iran. The results of the present study show that Karakul rams have continuous and acceptable spermatogenic activity during all seasons of the year. However, seasonal variations in semen characteristics are observed. Semen of superior quality was produced in late summer and throughout autumn.

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High ambient temperatures (average of 25 ◦ C), particularly in association with increasing day length during summer months has been demonstrated to result in a reduction in semen quality in rams (Perez et al., 1997; Karagiannidis et al., 2000). Southern Iran is regarded as a dry region with a hot summer (20–45 ◦ C). Contrary to our expectation, results of the present study show that semen output (volume × concentration) and semen mass motility of Karakul rams were not that much inferior in summer compared to that obtained in spring and winter. Similarly, Ibrahim (1997) in a study with Chios crossbred rams reared in the United Arab Emirates found that semen quality was not reduced during hot months of summer. Further libido of the Karakul rams (as defined by number of mountings and matings per 20 min) in summer were not significantly different to that of spring and winter. These findings suggest that the semen quality and libido of Karakul rams are not affected by summer high temperature in southern Iran. Semen could be collected successfully from the rams throughout the study. The mean semen volume, mass motility and the scrotal circumference of Karakul rams were significantly higher during autumn in the present study. This coincides with the maximum sexual activity of ewes of Karakul breed reared in most areas of southern Iran (Sefidbakht et al., 1978). The improvement in quality of Karakul ram semen during autumn (the breeding season) is consistent with the results of Colas et al. (1990) in Ile-de-France rams; Mandiki et al. (1998) in Suffolk rams and Karagiannidis et al. (2000) in Chios and Friesian rams. Further, a marked elevation in the mean plasma testosterone concentration was observed in coincides with the commencement of the breeding season (from late August towards November) in Karakul rams used in the current study. This finding is similar to the previous reports showing a significant rise in plasma testosterone concentrations in rams during the breeding season (Docchio et al., 1984; Rhim et al., 1993). Taking these findings and the current results into consideration, it is concluded that a high reproductive performance can be gained when Karakul rams are introduced to ewes during August till November. Scrotal circumference, a high heritable trait, is considered as an excellent index of sperm production in the ram (Toe et al., 2000). In the present study plasma

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concentrations of testosterone were highest at the middle and end of summer. Two months later, at the beginning of autumn, the scrotal circumference attained its maximum size. This relationship was also reported in other breeds of rams (Lincoln et al., 1990; Perez et al., 1997). The plasma concentration of testosterone decreased at the end of autumn and 1 month later the scrotal circumference decreased markedly. A similar pattern has also been reported in Merino rams reared on Australian grasslands (Bremmer et al., 1984) and in Corriedale rams under extensive management (Perez et al., 1997). A significant effect of season on scrotal circumference of Karakul rams was observed in the current study. This is in agreement with the results of Dufour et al. (1984) and Mandiki et al. (1998) who demonstrated a seasonal variation in scrotal circumference in Suffolk and Texel rams, respectively. The variation in scrotal circumference within temperate climates throughout the year seems to be due mainly to changes in photoperiod (Lincoln et al., 1990) and the effects are mediated by the pituitary gland and frequency of LH pulses (Rhim et al., 1993). Level of nutrition (Cameron et al., 1988) and daily body growth (Tulley and Burfening, 1983) have been shown to influence on scrotal circumference. The rams used in the present study were physically mature and they were effectively monitored not to fluctuate in their body weight during the study. Therefore, the changes in scrotal circumference could be attributed to the season and/or day length. The presence of the interaction between season and ram and also the observed individual variation in karakul rams are probably due to genetically unselected population from which six rams were obtained. These findings imply that selection of the genetically better Karakul rams is an important point to be borne in mind to obtain a high fertility.

5. Conclusions Results of the present study demonstrate that albeit Persian Karakul rams are reared under 40◦ latitude in the northern hemisphere they show seasonal fluctuations in their semen indices and scrotal circumference. However, the magnitude of the seasonal effects are not so marked to prevent the rams from being used for breeding purpose throughout the year. Semen of supe-

rior quality and quantity was collected in late summer and throughout autumn.

Acknowledgements The valuable assistance of Mrs. Z. Thaghipour and Kh. Moghadassi is highly appreciated. The authors wish to thank Mrs. M. Sharif for preparation of the manuscript.

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