Abstract: The present study were to evaluate the effect of individual bull, semen types, quality of bull semen, sources of semen on Non-return Rate (NRR) at 60 days of cows under field condition. A total 75550 cows were inseminated with 71 bull semens from Central Cattle Breeding Station and Dairy Farm (CCBSDF), Savar, Dhaka, Rajshahi Dairy and Cattle Improvement Farm (RDCIF), Rajabarihat and District Artificial Insemination Centre (DAIC), Rajshahi under 40 Artificial Insemination (AI) sub-centres/points of District AI centre, Rajshahi. The overall NRR was obtained 78.54% with chilled and frozen semen produced from three AI centres/stations. Analysis of variance showed that individual bull semen had significant (p<0.05) effect on NRR at 60 days after first insemination. Semen types, quality of bull semen and sources of semen had significant (p<0.001) effect on NRR at 60 days of cows. The significant (p<0.001) highest NRR (82.32%) was with chilled semen and lowest was with frozen semen (76.39%). The significant (p<0.001) maximum NRR (83.12%) was for the best quality bull semen and minimum (70.13%) for the poor quality bull semen. Significant (p<0.001) higher NRR (82.32%) was in semen from DAIC, Rajshahi and lower (73.01%) in semen from RDCIF, Rajabarihat. Results suggested that the NRR of cows at 60 days after first insemination under field condition may be a good practice to discard poor fertility semen among the individual bull semen, semen types (chilled and frozen), quality of bull semen (poor, good and best) and sources of semen (CCBSDF, Savar, RDCIF, Rajabarihat and DAIC, Rajshahi) for artificial insemination programme in Bangladesh.
INTRODUCTION
The frozen and chilled semen are very popular in the genetic improvement programme of livestock throughout the world. A review on Non-return Rate (NRR) with both types of semen seems that it maintains fertilizing potency even though fewer percentage of motile spermatozoa revive. The economic importance of higher breeding efficiency in dairy cows emphasizes the advantage of an accurate prediction of the post-thaw fertility of frozen semen. Evaluation of frozen semen has been based on a variety of test methods including estimation of motility defined as percentage of progressively motile spermatozoa, sperm velocity and acrosomic integrity (Saacke and White, 1972; Soderquist, 1991). The relationship between these parameters and fertility has been, however, disputed even though the NRR has been found to positively correlate with the percentage of post-thaw motile spermatozoa (Gibson and Graham, 1969; Linford et al., 1976). Frozen semen is useful and economic when bulls of superior index are exploited during a short period of life. This is also very economic in importing exotic germ plasm in the form of deep frozen semen straw rather than animal, which is very expensive. Numerous studies have described the effect of the bull or AI technician on female fertility (Humblot et al., 1991). Data suggests either little (Taylor et al., 1985) or large differences (Everett and Bean, 1982; Foote and Oltenacu, 1980; Jansen and Lagerwij, 1987; Williams et al., 1988) exist between AI bulls in conception ability. In France, early selection of young bulls for sperm production and libido was performed before progeny testing in dairy and beef breeds (Parez and Thibier, 1983). Season affects on NRR (Mahbub-E-Elahi et al., 1997). Performance of chilled and frozen semen with respect to NRR which has not yet been explored under field condition at greater Rajshahi district. The present study has been attempted to determine the effects of individual bull semen, semen types, quality of bull semen and sources of semen on NRR at 60 days after first insemination.
MATERIALS AND METHODS
The study deal with the evaluation of semen fertility under field condition by NRR at 60 days of cows using semen from various breeds of AI bulls. The study was conducted at three AI centres/stations and 40 AI sub-centres/points during the period from 1997 to 2002. The AI centres/stations were Central Cattle Breeding Station and Dairy Farm (CCBSDF), Savar, Dhaka, Rajshahi Dairy and Cattle Improvement Farm (RDCIF), Rajabarihat, Rajshahi and District Artificial Insemination Centre (DAIC), Rajshahi. A total of 75550 semen doses produced from 71 bulls at the three AI centres/stations were used absolutely for artificial insemination by 40 AI technicians at 40 AI sub-centre/points of DAIC, Rajshahi, Bangladesh. Seventy-one bulls among the six breeds of unknown field fertility, previously selected for reproductive performances and sperm production, were randomly included in this experiment. Forty AI sub-centres/points were selected out of 83 AI sub-centres/points under the DAIC, Rajshahi on the basis of better AI performances. Forty AI technicians of the respective AI sub-centres/points performed AI. All Field assistants (Artificial Inseminator) were government employees and had one year training on AI. Each Field assistant had done 90-450 AI/month regularly. All inseminators at the 40 AI sub-centres/points were considered equivalent. Finally, a total of 4 districts (Naogoan, Natore, Nawabgonj and Rajshahi) with 40 (20 of chilled and 20 of frozen semens) AI sub-centres/points was included in this study.
Data collection and record keeping: In July 1997, selected technicians (Field Assistant, Artificial Inseminators) from different AI sub-centres/points of the 4 districts were brought at District AI centre, Rajshahi, for training on fertility of AI bulls. Assistant Director, DAIC, Rajshahi, organized the programme. The training programme covered the topics on semen, AI and how to determine the Non-return Rate (NRR) at 60 days. Researchers made a special format for accurate data record for NRR and supplied the formats among the 40 technicians. The information, such as date of AI, semen types, bull ID, breed, semen production date, month of AI, name of AI centre and sperm motility percentage, date of return for repeated AI after the first insemination etc. were included in this format.
Individual bull semen: Out of 71 bull, 50 bulls were from CCBSDF, Savar, Dhaka, 7 from RDCIF, Rajabarihat, Rajshahi and 14 from DAIC, Rajshahi. All the bulls were produced from CCBSDF, Savar and distributed into RDCIF, Rajabarihat, Rajshahi and DAIC, Rajshahi on the basis of indent. Bull ID/name of bull was given by Central Artificial Insemination Laboratory (CAIL) at CCBSDF, Savar, Dhaka, Bangladesh on the basis of breeds of bull viz. pure breed, cross-bred, local breed, sources of bull/semen, pedigree of bull etc. The management systems of these bulls were maintained as per recommendation of CCBSDF, Savar, Dhaka. Feeding and management system in three AI centres/stations were more or less uniform throughout the experiment. All the bulls of the AI centres/stations were kept in similar housing and management. The bulls were housed individually in pens with sufficient cross ventilation and protected from hot-sun and heavy rainfall to avoid abrupt fluctuation of their body temperature during the whole period. The bulls were exercised once in a week in the form of running on the field. No bull was affected with remarkable diseases. The bulls of three centres were regarded as clinically healthy and free from any significant abnormalities. The bulls were vaccinated routinely against Anthrax, HS, Blackleg, FMD and dewormed against Fascioliasis and Roundworm.
Semen types, Semen processing and preservation procedure: Two types of semen (chilled and frozen) were produced from 3 AI centres/stations and used randomly for AI at greater Rajshahi.
Chilled semen: District AI centre, Rajshahi, routinely used Egg yolk- citrate extender for processing of chilled semen. The semen was diluted to obtain 20x106 progressive motile spermatozoa per ml i.e., an insemination dose. Individual insemination doses were transferred into glass vial, covered by cork and preserved at +4 to 8°C until used. Semen showing less than 50% motility after processing was not preserved for AI.
Frozen semen: For frozen semen at Central AI laboratory, CCBSDF, Savar, Dhaka and RDCIF, Rajabarihat Egg-yolk Tris- fructose acid glycerol extender were used routinely. The semen diluent contained 6.4% (v/v) glycerol and 30x106 spermatozoa per 0.25 mL i.e., one individual dose. Individual insemination doses were succeed in 0.25 mL French straws and the straws were preserved in liquid nitrogen (-196°C) in cryocan. Only semen straw showing that ≥40 % post thaw motility was preserved for AI.
Gradation of semen quality: The bull semens were graded as best, good and poor quality based on pre-set criteria on semen evaluation data given in Table 1.
If semen characteristics from a bull did not meet any one or more criteria mentioned in the above table, it was classified as good or poor. The values set in the criteria were the mean of 5-10 sample of individual bulls. The gradation of bulls was completed before analyzing fertility data.
| Table 1: | Preset criteria for the gradation of bull semen |
Sources of semen and semen supply: A total of 27,398 chilled semen doses were used for AI from 14 bulls at DAIC, Rajshahi and 48,152 frozen semen doses for AI from 57 bulls (39,356 from CCBSDF, Savar and 8,796 from RDCIF, Rajabarihat at deep frozen semen production laboratory). Both types of semen, chilled and frozen, were labeled clearly such as Bull ID, breed, production date, name of AI centre and sperm motility percentage. The bulls, which performed at least 200 AIs were taken into account. In the present study, the number of first AIs varied between 200 and 3276 per bull. Two types of semen were used randomly for AI at greater Rajshahi. All the semen doses were recorded and semen of each bull was then despatched at random among 40 AI technicians of respective AI sub-centres/points in the quantities needed. Semen preservation and transportation were maintained as per national cattle breeding programme instructions.
Oestrus detection and insemination of cows: At first, the farmers detected oestrus of cows on the basis of the following signs and symptoms: stand to be mounted, bellowing, restlessness, mounted to others and mucus discharge from vagina. Then the cows were brought to AI sub-centres/points for AI. Finally, AI technicians confirmed the detection of oestrus of cows after clinical examination. Information like name and address of owner of cows, cow history, semen data etc. were recorded in AI register and AI card was issued to client after performing AI. The cows were inseminated transcervically by recto-vaginal method (Olds, 1978) with the help of inseminating gun for frozen semen and plastic AI tube pipette for chilled semen. The cows were inseminated within 12-18 h of oestrus. The AI technicians instructed after AI the owner of cow to inseminate by the same AI technicians if the cows were not conceived within 60 days. The NRR at 60 days after insemination was subsequently calculated from a total of 75,550 first AIs. The Non-return Rate (NRR) was calculated by using the following formula:
Statistical analysis: The analysis data of NRR at 60 days was carried out by non-orthogonal analysis of variance (ANOVA) with the MODEL programme (Kobilinsky, 1980); each non-return rate 60 days post AI was weighed by the number of first AI’s from which it was derived. The general analysis on the whole set of data was made with the following fixed model:
Zijkl = μ+Ai+Bj+Ck+Dl+Eijkl
Where,
Zijkl represents the individual non-return rate at 60 days
Ai is the effect of individual bull
Bj is the effect of types of semen
Ck is the effect of quality of bull semen
Dl is the effect of sources of semen and
Eijkl corresponds to the random error associated with Zijkl.
The effect of each factor was tested after adjustment for the others. The statistical differences between 60 days NRR and each treatment group were subsequently analyzed by Chi-square test.
RESULTS
Non-return rate (NRR) is a good, rapid, inexpensive method to compare fertility of bulls, efficiency of AI centres/stations as well as to compare fertility among herd of the cows. A total of 75550 inseminated cows with different types of semen from 71 bulls belonging six genetic groups at 40 AI sub-centres/points among three AI centres/stations were studied during the experimental period of 5 years. Effects of individual bull semen, types of semen, quality of bull semen and sources of semen on NRR under field condition are presented in (Table 2-4).
Individual bull semen effect: The study evaluated the effect of 71 bull semen on 75550 cows for NRR at 60 days. The results on the NRR at 60 days in cattle due to artificial insemination with chilled and frozen semen bulls under field condition have been presented in Table 2.
| Table 2: | Non-return rates in cattle due to artificial insemination
with chilled and frozen semen from individual bulls under field condition |
| NRR = Non-return rate, CCBSDF = Central Cattle Breeding Station and Dairy Farm, RDCIF = Rajshahi Dairy and Cattle Improvement Farm, DAIC = District Artificial Insemination Centre | |
| Table 3: | Semen types, quality of bull semen and sources of semen effect
on non-return rates at 60 days after first insemination of cows under field
condition |
a,b,cData in column with different superscripts
are significantly different from each other(p<0.05). CCBSDF = Central
Cattle Breeding Station and Dairy Farm, RDCIF = Rajshahi Dairy and Cattle
Improvement Farm, DAIC = District Artificial Insemination Centre |
|
| Table 4: | Analysis of variance for determining individual bull semen,
semen types, quality grade of bull semen and sources of semen effect for
non-return rates at 60 day of cows after first insemination |
| * = p<0.05; *** = p<0.001 | |
The NRR varied from 65.39 to 87.52% depending on bull. The overall mean NRR was obtained 78.54% with chilled and frozen semen. Analysis of variance showed that individual bull had significant (p<0.05) effect on NRR (Table 4). The highest NRR was for bull ID # 163 (87.52%) in DAIC, Rajshahi followed by for bull ID # 5848 and 4414 (85.27 and 85.21%, respectively) (Table 2). The lowest NRR (65.39%) was obtained for bull ID # 7707 at RDCIF, Rajabarihat and followed by bull ID # 3498 and 40253 at CCBSDF, Savar for frozen semen (66.81 and 67.00%, respectively) (Table 2).
Semen types, quality of bull semen and sources of semen effect: In the statistical analysis, NRR at 60 days of cows were significantly affected by the interaction between semen types, quality of bull semens and sources of semen (p<0.001). The effect of semen types, quality of bull semen and sources of semen on NRR are presented in Table 3. Semen types, quality of bull semen and sources of semen had significant effect on NRR. The analysis of variance indicated significant difference for the NRR by different factors (Table 4). The significant highest NRR (82.32%) was found with chilled semen and lowest (76.39%) with frozen semen of semen varieties. The significantly highest NRR (83.12%) was found in best quality bull semen and lowest (70.13%) in poor quality bull semen. The higher NRR (82.32%) at 60 days was found in semen from DAIC, Rajshahi and lower (73.01%) in semen from RDCIF, Rajabarihat. The overall mean non-return rate was obtained 78.54% with two types of semen which were produced from 3 AI centres/stations.
DISCUSSION
In the present study, individual bulls differed with regard to the NRR at 60 days of cows. The differences between bulls may be attributed to the variation in the secretory activity of sex glands, scrotal circumference, breed, age, body size and body weight (Graves, 1978; Leon et al., 1991; Sharma et al., 1991). With respect to semen quality more abnormal spermatozoa were found in older bulls (Rao, 1971). Similarly, higher post-thaw motility of spermatozoa was found in younger bulls (Foote, 1986). Age related variation in the quality of semen may be due to differences in the composition of the seminal plasma. The breed of bulls can influence the quality of preserved semen with regard to post-thaw motility and proportion of abnormal spermatozoa (Bhupal et al., 1993; Tegegne et al., 1994). The breed related variation in the semen quality can also be attributed to the difference in the composition of seminal plasma (Mohan et al., 1994; Nandroo et al., 1987). Therefore, it is likely that the variation in the semen in the present study may be due to differences in the age and breed of bulls. This means that either bull of DAIC, Rajshahi are more fertile or chilled semen contained more effective viable spermatozoa than deep frozen semen. A combination of these two factors might also be possible. The results differ from Humblot el al. (1991) who noted higher NRR in Holstein bulls than in Normad and French Friesian. Gerard (1991) observed that the average NRR at 60-90 days post AI for the whole experiment was 67.95% which differs with present findings. The variation may be due to the length of NRR and the experiment conducted at field condition. Gerard (1991) also observed that individual bull and AI centres have significant effect on NRR at 60 to 90 days (Gerard, 1991). These results agree with those of Jansen and Lagerwij (1987) who found 10% difference in terms of fertility due to the AI sub-centres/technicians. The individual bull, semen preservation method, mode of semen transportation were found as the most important factors of variation of NRR at 60 days.
The data of the present study revealed that NRR was significantly higher of cows inseminated with chilled semen than those inseminated with frozen semen. In general, the NRR after insemination with chilled semen is higher than that obtained after insemination with frozen semen, provided the preservation temperature remains constant (Coulter, 1992; Foote, 1986; Shamsuddin et al., 1987). It is likely that freezing and thawing procedures damage some spermatozoa in an insemination dose and thereby decreases the number of viable spermatozoa (Coulter, 1992; Shamsuddin and Rodriguez-Martinez, 1994). Owing to the larger volume of chilled semen dose compared to frozen ones, the chilled semen has an additional merit of rapid and easy dispersion of spermatozoa in female genital tract. Fertility data retrieved from routine AI with chilled or frozen semen are contrasting. For instances, the conception rate after insemination with frozen semen were slightly lower than those achieved with chilled semen (Coulter, 1992; Foote, 1986; Shamsuddin et al., 1987). The lower fertilizing capacity of spermatozoa in frozen semen may be attributed to damage or injury to the most spermatozoa during freezing and thawing resulting in decreased number of viable spermatozoa (Coulter, 1992). However, Nadir et al. (1993) did not find difference in fertility between inseminated with fresh (+4 to 5°C) and frozen semen in straws.
The significantly highest NRR (83.12%) was in best quality bull semen and lowest in poor quality bull semen (70.13%). Bhuiyan et al. (1999) reported that cows inseminated with good quality bull semen (having >50% motility, >7.5x106 total number of motile spermatozoa and >70% normal sperm for chilled and >80% normal sperm for frozen) conceived at higher rate than those inseminated with poor bull semen (55.2% vs 37.1%, p<0.001). The variation in the NRR between the best and poor quality semen in the present investigation may be due to the fact that best quality bull semen contained higher proportion of morphologically normal motile spermatozoa than that of poor quality semen. It is well documented that the fertilizing capability of spermatozoa depends on the innate fertilizing capacity of bulls as well as deposition of optimum number morphologically normal spermatozoa into the uterus in time (Saacke et al., 1991). Gradation of bull semen quality on the basis of pre-set criteria related to the number of morphologically normal viable spermatozoa may be used as predictor for fertility. The variation in the quality of preserved semen may be due to man made manipulation or to innate fertility of individual bulls. Man made deterioration in the quality of semen may occur during semen collection, processing, storage and transportation. Too frequent collection or prolonged sexual rest can produce poor quality semen characterized by increased frequency of abnormal spermatozoa which in turn may reduce fertility (Amann, 1986; Sullivan, 1978; Watson, 1990).
The NRR was significantly higher after insemination with frozen semen from CCBSDF, Savar, Dhaka than that after insemination with locally produced frozen semen of RDCIF, Rajabarihat, Rajshahi. Also, the locally frozen semen had more damaged spermatozoa than the frozen semen of CCBSDF, Savar, Dhaka. This indicates insufficiency in the refinement of freezing technique. Improper semen freezing technique is one of the major causes of fertilization failure (Parks and Graham, 1992). Further, the frozen semen of CCBSDF was used mostly in cross-bred dairy cows with good health condition and inseminated by more efficient technicians. These extrinsic factors perhaps positively influenced the NRR in favour of frozen semen of CCBSDF, Savar, Dhaka. The higher NRR (82.32%) at 60 days was found in semen from DAIC, Rajshahi and lower (73.01%) in semen from RDCIF, Rajabarihat. Gerard and Humblot (1993) reported significant (p<0.05) the effect of AI centre was on NRR. The variation observed in NRR among sources of semen could be due to variation of assessment in recording observation, fertility of inseminated cows, distance of AI sub-centres/points from DAIC, Rajshahi, preservation system, skillness of FA (AI) and mode of transportation.
From the experiment the following conclusion can be drawn i) NRR of cows at 60 days after first insemination may be a good practice to discard poor fertility bull semen batch from AI programme ii) Bull ID #: SL-163, SLxF-5848 and LxF-4414 had good fertility rate (NRR at 60 days) iii) NRR with the chilled semen had good fertility than the frozen ones iv) The best quality bull semen had better NRR at 60 days of cows after first AI and v) Bull semen of District AI centre, Rajshahi yielded higher NRR at 60 days of cows.