The present study is designed to find out normal values of major biochemical constituents of seminal plasma, reproductive parameters and effect of age on semen quality and quantity of Cholistani and Sahiwal bull semen. For this purpose 42 experimental bulls (eleven years old bulls and ten years young bulls of the each breed) will be selected and maintained at the Semen Production Unit, Karaniwala, Bahawalpur. The bulls will be divided into three different groups according to age; semen will be collected from all the bulls twice a week with standard artificial vagina method. Immediately after collection, semen will be shifted to water bath at 37 ËšC and subjected to physical tests, i.e., volume, color, mass motility, motility percentage, concentration of spermatozoa, staining for morphological abnormal spermatozoa and differential staining of live and dead spermatozoa. Furthermore, seminal plasma will be separated by centrifugation of semen samples at 3000 G for 20 minutes. The supernatant will be separated and stored at -20 ËšC till analysis of biochemical constituents. Other reproductive parameters of these bulls (scrotal circumference, libido characteristics) and body conditions will also be studied. The data thus obtained will be analyzed by correlation coefficient and regression analysis.
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Agriculture is the mainstay of Pakistan's economy and Livestock sector is one of the major pillars of agriculture that plays important role in the economy of Pakistan by providing essential food items like milk, meat, eggs, hides, skins and wool. Livestock contributed approximately 55.1 percent to the agricultural value added and 11.6 percent to national GDP during 2010-12, against 54.6 percent and 11.6 percent during the same period, last year. The total cattle population in Pakistan is 36.9 Million heads, producing 16,741 thousand tons of milk (Pakistan Anonymous, 2011-12).
Cholistani and Sahiwal cattle breeds are very important due to their resistance to harsh environment and reasonable production at optimum level. Sahiwal breed is well known among Zebu cattle for its superior dairy qualities. It has been exported to many countries for both cross and pure breeding purposes (Maule, 1990). Cholistani is a multi-purpose breed, being used for both meat and milk and as a draft animal (Mason, 1996).
Low fertility is generally considered to be a primary constraint to cattle production in tropical regions, where most of the animals are of Zebu (Bos indicus) breeds (Chenoweth, 1991; Vandeplassche, 1982). The productive and reproductive performance of cattle can be enhanced by selective breeding. In this context, artificial insemination (AI) is a powerful biotechnological tool that allows producers to use superior sires for promoting faster genetic improvement and increasing profitability (Brito et al., 2002). In Pakistan, about 4% buffalos and 14% cattle are bred with AI and the rest are bred with natural mating or not bred due to shortage of breeding bulls (Ahmad and Saji, 1997). Reproductive performance of bull is of particular importance among the parameters affecting fecundity and reproductive efficiency of a herd (Boyd, 1991; Vandeplassche, 1982); however, limited information is available on this subject (Galina and Russell, 1987, Galina and Arthur, 1991). Though, studies have been conducted on Bos taurus breeds but Bos indicus breeds remain to be under- studied in this regard (Silva et al., 2000).
Moreover, in dairy breeding, artificial insemination has key role in reproductive health and profitability. Artificial insemination is a biotechnological tool of prime value for proper genetic selection and breeding hence it offers the possibility for upgrading economic output of dairy farming. Quality of semen plays a vital role in the success of breeding plan for improving dairy cattle through artificial insemination. The characteristics of bull semen vary widely not only between bulls but also within the bull from time to time and season to season (Nadaraja, 1967).
Reproductive ability in the male comprises of the production of semen containing normal spermatozoa (quality) in the adequate number (quantity), together with the desire and ability to mate. Sperm output is an important index for judging the functional capacity of testes in breeding bulls. Wide variations in sperm output capabilities exist among breeds and among individual bulls within a breed depending upon their genetic potential, collection procedure and many other environmental and management factors (Amann and Almquist 1962, Salisbury et al., 1985, Tuli et al., 1988). Semen quality evaluation is determined by: macroscopic (volume, color, odor, and consistency) and microscopic (motility, morphologic properties, and sperm concentration) analysis. Anyhow, recent research proves that employing this as the only parameter of semen evaluation is not sufficient because semen proven good by such evaluation may lead to fertility issues. Therefore, apart from conventional method of semen analysis involving estimation of mass motility, sperm concentration and morphology of sperms; assessment of some other biochemical constituents has been suggested (Argov et al., 2007, Turba et al., 2007, Brinsko et al., 2007, Meseguer et al., 2004).
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The seminal plasma is a complex mixture of secretions from the epididymyds and various accessory sex glands, which provides the medium and vehicle for spermatozoal survival. Seminal plasma contains a variety of biochemical components, some of which are relatively specific for the regulation of sperm function (Gundogan, 2006). The composition of seminal plasma is determined by the size, storage capacity and secretory output of different organs of the male reproductive tract (Mann, 1964). Evaluation of these biochemical factors is an important criterion for assessing male fertility. Deviation from normal values of these biochemical components in seminal plasma is correlated with male infertility (Cevk et al., 2007).
Biochemical estimates of seminal plasma are used for semen evaluation as seminal plasma play its role in sperm metabolites, nutrition of ejaculated sperms and provides protection to spermatozoa against proteinase inhibitors, which help in sperm capacitation and local immunosuppression (Pesch et al. 2005). Seminal plasma comprises of ions (Na+, K+, Zn+, Ca++, Mg++, C++); energy sources (fructose, sorbitol, glycerylphosphocholine); organic compounds (citric acid, amino acids, peptides, low and high molecular weight proteins, lipid, hormones, cytokines); and nitrogenous components such as ammonia, urea, uric acid and creatinine. Reducing substances such as ascorbic acids and hypotaurine also exist in the seminal plasma of ruminants.
The present study is being conducted with the following objectives:
1. To compare the basic levels of biochemical constituents in seminal plasma of Cholistani and Sahiwal bull.
2. To compare the spermiogram of Cholistani and Sahiwal breeds.
3. To compare the libido parameters of Cholistani and Sahiwal breeds and relate their effects to quality of the semen.
4. To find out the effect of age on the quality and quantity of semen in Cholistani and Sahiwal bull.
REVIEW OF LITERATURE
EFFECT OF AGE AND SEASON ON SEMEN QUALITY
Brito et al., (2002) studied 11 Bos taurus and 7 Bos indicus bulls at artificial insemination (AI) center, Brazil. The effects of ambient temperature, humidity, month and age on quality of semen and production of sperm were evaluated. Data from two consecutive years were analyzed separately. The quality of semen and production of sperm was not affected by ambient temperature and humidity probably because there was little variation in these variables. Increased bull age was associated with decreased sperm motility (P< 0.10) and increased minor sperm defects (P< 0.001). B. indicus bulls had greater (P< 0.005) sperm concentration than B. taurus bulls in both years (1.7 - 109/ml versus 1.2 - 109/ml in Year 1 and 1.6 - 109/ml versus 1.2 - 109/ml in Year 2, respectively).
Vilakazi and Webb (2004) studied 271 Friesland artificial insemination bulls of different ages in South Africa for five years. Semen was collected with artificial vaginaand analyzed for major and minor defects and also for normal sperm cell percentage. The results showed that the age of bull had effecton major sperm defects and normal sperm percentage. Mature bulls of age 36-48 month exhibited less sperm morphological defects than older (more than 72 month) and younger (less than 36 month). Major defects of sperm due to age were knobbed acrosomes, dag defects, mid piece reflexes and pyriform head. The season also affected the normal sperm percentage significantly. The sperm major defects were recorded as 11.5±2.0%, 20.3±1.3%, 15.3±1.9% and 12.5±2.0%; and normal sperm percentage as 84.4±2.4%, 72.8±1.6%, 79.4±2.2% and 82.5±2.4% for spring, summer, autumn and winter, respectively. Sperm morphology also varied with season of the year as better spermiogram was observed in winter and spring than in autumn and summer. Moreover, major sperm defects were affected by the season and age interaction as in older bulls prevalence of major sperm defects was higher in summer and in young bulls higher in winter season.
MINERALS ANALYSIS FROM THE SEMINAL PLASMA
Cragle et al., (1958) reported that potassium is more concentrated within the sperm cells of bovine semen than in seminal plasma. But the sodium and calcium are more concentrated in the seminal plasma than within the sperm cells. In seminal plasma the different levels of potassium and sodium were different in individual bulls and in different ejaculates from the same bull when consecutive ejaculates were analyzed. They found, increasing sodium and chloride concentrations and decreasing potassium and calcium concentrations, as increasing consecutive individual ejaculates were analyzed.
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Igboeli and Rakha (1971) used flame photometer for potassium and sodium concentration and atomic absorption spectrophotometer for magnesium and calcium in whole semen and seminal plasma. Sodium concentration (mg/100 ml) in seminal plasma and whole semen and the fraction of pre ejaculate was 320±9.9, 347±8.4 and 335±47.1, respectively while in the same order potassium was 69.4±3.1, 71.4±4.5 and 152±37.8; Ca was 34.0±1.4, 35.3±1.1 and 4.1±1.4 and Mg++ was 8.8±0.06, 8.3±0.3 and 5.7±1.7.
Holtz and Foote (1978) calculated correlation among different seminal constituents. Semen was collected twice a day at alternate days from 12 Dutch belted rabbits. Sexual stimulation, ejaculate volume, appearance and sperm counts was observed. Seminal plasma was analyzed for biochemical contents like fructose, citric acid and minerals (Na, K, Mg, Ca and Zn). Second ejaculate had lesser concentration of fructose, citric acid, K and Zn than the first ejaculate. Fructose was formed in the different portions of the prostate gland and major concentrations of citric acid were added in ampulla of the ductus deferens and also by vesicular gland.
Kavanagh (1985) reviewed that ionic composition of human prostatic fluid varied among individuals. He reported that major ion was citrate, in normal prostatic fluid, while chloride concentration was lower. Counter ions for these were mainly potassium and sodium along with zinc, calcium and magnesium. Citrate was associated with pH, as it increased amount lead to decreased pH.
Wong et al., (2001) concluded that zinc, copper, calcium and magnesium in seminal plasma and blood affected spermiogram. Semen and blood specimens were collected from 103 sub fertile and 107 fertile males. Total magnesium and calcium concentrations were calculated by colorimetric end point assay. Flame atomic absorption spectrophotometer was used for copper and zinc analysis. Zinc, copper, calcium and magnesium concentration in seminal plasma and blood did not differ in fertile and sub fertile. Correlation was found between zinc concentration in blood plasma and abnormal sperm morphology (rs = 0.13), sperm count (rs = 0.18), and sperm motility (rs = 0.15); and blood plasma copper concentration was correlated with motility (rs = 0.25). While seminal plasma´s magnesium and zinc concentrationswere correlated with sperm count (rs = 0.16 and rs = 0.17). Between zinc, calcium and magnesium strong correlation were found in seminal plasma. They argued that for fertility and spermatogenesis: seminal plasma concentrations of copper, zinc, calcium and magnesium are very important.
Mosaferi et al., (2005) investigated seminal characteristics in Bactrian camel. Semen samples were collected with modified bovine artificial vagina from 10 mature camel bulls. Biophysical parameters of sperm like concentration, volume, progressive motility, color and percentage of live and dead spermatozoa were measured. Moreover, biochemical parameters like total protein, calcium, chloride, phosphorus, glucose, phospholipids, osmolarity, albumin, triglycerides, non-protein nitrogen concentrations in seminal plasma and seminal pH were also measured. The semen volume varied from 1.2 to 26 (average 8.2± 0.7 ml). Color and consistency of most semen samples were milky; average semen osmolarity was 316.1 ± 1.48 mOsm/kg H2O; semen pH was basic (7.4 ± 0.03); sperm mean concentration was 414.8 ± 25.04 x106 cells/ml; spermatozoa progressive motility was 62.4 ±1.57% and live spermatozoa percentage was 85.6 ±1.15.. Concentration of glucose, total protein, albumin and non-protein nitrogen were measured as 35.8 ± 0.9 mg/dl, 32200 ± 100 mg/dl, 31100 ± 100 mg/dl and 32.5 ± 2.5 mg/dl, respectively. In seminal plasma the triglycerides and phospholipids concentrations were 101.6 ± 5.5 mg/dl and 8.2 ± 0.1, 2.9 ± 1.7 mg/dl, respectively. Chloride, calcium and phosphorus concentrations were 97.9 ± 2.9 mEq./l,8.2 ± 0.1 mg/dl and 2.9 ± 1.7 mg/dl respectively.
Janicki et al., (2008) studied 45 samples of bull semen for Zn and Pb concentration to evaluate the correlation between zinc, lead and other parameters that affect spermiogram and seminal characteristics. Atomic absorption spectrophotometer (AAS) was employed to measure zinc and lead. Significant positive correlation was found between sperm motility, ejaculate volume and zinc concentration. Lead concentration and the analyzed parameters were not correlated at all.
Valsa et al., (2008) conducted a study to find out if any, within subject variability, exists in semen parameters, levels of magnesium and calcium in seminal plasma and spermatozoa. They found significant changes in semen volume and in slowly moving sperm cells fraction. But, other seminal constituents and both magnesium and calcium, either within spermatozoa or in seminal plasma, did not show any significant variation.
Lukac et al., (2009) evaluated zinc, manganese, copper, iron, nickel, cadmium and lead concentrations in rabbit semen and their relation to quality of spermatozoa using atomic absorption spectrophotometer. Semen samples were stained with Giemsa dye and fixed in Hancock's solution and almost 500 spermatozoa were evaluated for each rabbit. In rabbit semen trace elements concentrations were estimated as follow zinc=81.20±59.43 mg/kg, nickel=0.297±0.11 mg/kg, iron=19.65±7.26 mg/kg and copper=20.10±4.09mg/kg, along with a total 16.21±7.99 % pathological of spermatozoa.