Methods of preserving bull semen
AI application in developing countries
Conclusions
Bibliography
H. SchuhThe author was an associate professional officer in the Animal Production
and Health Division, FAO. His address is Stöckacher Weg 6, Birkenfeld,
D-8530 Neustadt/Aisch, Germany.
Artificial insemination (AI) is a globally accepted method of breeding cattle and is also effective for other species. An estimated worldwide total of 150 million cows are artificially inseminated, while the number of artificially inseminated females of other species is uncertain (Bonadonna and Succi, 1980).
AI shares the same advantages and disadvantages in both developed and developing countries, but the cost-benefit factors are quite different. The transportation of inseminators and their equipment all the year round is a basic major cost in the management of this technique and can sometimes become prohibitive for a successful AI operation in developing countries. Further difficulties are a lack of support services such as telephone communication, electricity, equipment, maintenance and the supply of spare parts. These factors should therefore be taken into consideration when AI is to be applied in developing countries. The technique should fit into a given infrastructure and be adapted to available facilities.
Herd size in developing countries is generally small, the use of herd bulls is costly and the spread of reproductive diseases by natural service can be a danger. From this viewpoint alone, not to mention its immense potential in animal breeding, AI is to be preferred.
In most of the developing countries, AI was introduced on a small scale during the 1950s and 1960s and was carried out with fresh or room-temperature (RT) semen. During the late 1970s deep-frozen semen started to be processed while donor agencies encouraged the introduction of highly specialized and costly AI establishments, supported by international investment but with little thought given to the prospects of their maintenance. In view of this, operational capability still depends in many cases on the availability of donor funds. The advantages of long-term storage have therefore convinced producers in developing countries to practice AI with frozen rather than liquid semen.
Generally, the foremost advantage of AI is that it can be carried out independently of the sire's presence. Furthermore, it allows an enormous multiplication of outstanding genetic material and serves as a prophylaxis against spreading venereal diseases. Semen can be stored deep-frozen in liquid nitrogen (LN2), in a chilled liquid form or at room temperature. In terms of costs, the production of frozen semen requires a much higher investment in laboratory equipment while the necessary machinery and instruments incur heavy maintenance costs.
The method of preserving semen in a liquid form allows an intermediate storage of a few days only while the deep-frozen form permits storage for years without any significant decrease in semen quality. According to the length of storage planned, different semen extenders have been developed.
The composition of a semen extender is mainly based on an energy resource (sugars such as glucose and lactose) and a buffer medium of different inorganic or organic salts. Milk and egg yolk, for example, are basic ingredients of most extending media (Salisbury, van Demark and Codge, 1978). Egg yolk, especially, is recognized as a protectant against cold shock through its lipoprotein and phosphatidylcholine (Evans and Setchell, 1978). Semen extender solutions of approximately 20 percent egg yolk have become standard for use in most extenders. In the case of deep-frozen semen, glycerol is added for cryopreservation and it remains the standard cryopreservative agent. When semen is frozen by conventional methods in a glass ampoule, approximately 7 percent of glycerol is optimal for egg yolk citrate (EYC) and TRIS extender, 11 to 13 percent for fresh and reconstituted skim milk (Salisbury, van Demark and Codge, 1978). The addition of glycerol may enhance motility, though the advantage has generally been small (Foote, 1970).
The average sperm production of a bull ranges between 3.6 and 12 x 109 sperm per ejaculate. When using liquid semen, a minimum amount of 2.5 million total sperm per dose is required. Consequently, each ejaculate could provide 1 440 to 4 800 doses. However, when using frozen semen, approximately 20 million total sperm per dose are required. This would amount to 180 to 600 doses of frozen semen per ejaculate.
In principal, thawing solutions follow the same requirements as the semen extender. In 1964, Nagase, Graham and Niwa (cit. Omer, 1971) reported the influence of different thawing solutions on the fertility of pellet semen (see Table 1). Of the solutions tested, buffered glucose milk showed the best results. However, in this case the storage time is usually very limited after thawing.
1 Thawing solutions for pellets and the fertility rates achieved
Solutions de décongélation des paillettes et taux de fécondité obtenus
Soluciones de descongelación de pastillas, con las tasas de fecundidad conseguidas
|
Solution |
Fertility rate (%) |
|
Buffered glucose milk |
78.1 |
|
Minnessota Go |
77.8 |
|
Milk |
76.0 |
|
3% sodium citrate (pH 7) |
73.5 |
|
TRIS buffer |
71.4 |
|
0.9% sodium chloride |
70.0 |
|
10.5% lactose |
69.3 |
|
3% glycol (pH 7) |
69.2 |
|
Coconut milk glucose (CMG) |
77.6 |
|
Hannover thawing solution (HTS) |
80.0 |
Source: Omer, 1971.
Shannon (1968) made a trial to test the effect of oxygen levels on the survival of sperm. Split ejaculates from 20 bulls were stored at 5°C at concentrations of 12.5 and 200 million sperm/ml in egg yolk citrate (EYC) diluents, modified by the addition of glycine and glycerol (14G), and by glycine, glycerol and caproic acid (in the case of 14GC). At days two, six and eight of storage, samples were incubated at 37°C. Samples containing 200 million sperm/ml and stored at 5°C were rediluted in the appropriate diluent to contain 12.5 million sperm per ml, immediately before incubation.
The results of this trial are shown in Table 2. The three principal features of the trial were: significant differences in survival according to dilution rates; significant interaction between nitrogen saturation and dilution effects; and significant interaction between nitrogen saturation, days of storage and dilution rates.
2 Incubated life (hrs)1 of sperm stored at 37°C at 12.5 million and 200 million sperm/ml
Durée de vie en incubateur de spermatozoïdes conservés à 37°C à des concentrations de 12.5 millions et de 200 millions/ml
Vida en incubación a 37°C de semen almacenado a concentraciones de 12,5 millones y 200 millones de espermatozoides/ml
|
|
Unsaturated diluents |
N2-saturated diluents |
||
|
(14G and 14GC) |
(14G and 14GC) |
|||
|
Storage concentration |
||||
|
Days of storage |
12.5 |
200 |
12.5 |
200 |
|
|
(million/ml) |
|||
|
2 |
39.9 |
45.0 |
47.6 |
51.2 |
|
6 |
31.9 |
42.1 |
48.3 |
50.0 |
|
8 |
23.0 |
37.3 |
47.9 |
50.5 |
|
Average |
31.6 |
41.5 |
47.9 |
50.5 |
Note: All samples were initially incubated at 12.5 million sperm/ml.1 Assuming the end of life to be the time at which 10 percent of the sperm were still motile, as cited by Shannon and Curson (1984).
The effect of nitrogen saturation, therefore, was to halt the decline in incubation life associated with ageing semen, and this effect was much more marked for semen stored in a highly diluted form compared with semen stored in a concentrated form. It seems unlikely that this effect is caused by nitrogen as such, but rather by a reduction of oxygen tension in the media.
One of the products of aerobic metabolism in bull sperm is peroxide. This has detrimental effects on survival and is produced by the dead sperm enzyme aromatic-L-amino acid oxidase. It can be eliminated effectively by the addition of catalase to the diluent. Catalase improves sperm survival even in nitrogen-saturated diluents, and a combination of nitrogen saturation and catalase addition is a most effective form of protection against dilution effects at very high dilution rates (Shannon, 1968). Thus, in the case of RT semen (Coconut milk extender, CME, and Caprogen extender) catalase was added to neutralize hydrogen peroxide in the media.
As egg yolk provides the substrate for the dead sperm enzyme aromatic-L-amino acid oxidase, lowering the concentration of egg yolk in the diluent will also reduce the final concentration of hydrogen peroxide. Summarizing activity can be reduced by: lowering the concentration of egg yolk, which provides the substrate for this enzyme; and lowering O2 tension in the diluent at 30 and 40 percent air saturation of diluents. The subsequent activity of this enzyme is, respectively, 40 and 55 percent of its activity at 100 percent air saturation (Shannon and Curson, 1984).
To test the effect of catalase additions, a trial was conducted by Shannon (1968), who compared the effects of different dilutions with and without catalase and at two different levels of egg yolk (see Table 3). There were two main results: first, the conception rate did not decline despite higher dilution rates; second, although the addition of catalase appeared to give improved conception rates, it apparently did not affect the dilution rate response. Consequently, through improvement of the Caprogen diluent, the number of sperm per insemination has been progressively reduced from five million total sperm to two million total sperm per insemination (Shannon, Curson and Rhodes, 1984).
A series of trials was conducted by Shannon and Curson (1984) to determine the optimum storage temperature for sperm diluted in Caprogen. Survival of sperm at 37°C in vitro was significantly higher after storage at 16 to 20°C than after storage at 5°C (p<0.001) and also significantly higher after storage at 15.6 to 21.1°C than at 26.7 or 32.2°C (p<0.001).
To lengthen storage of rediluted pellet semen, Omer (1971) modified the Cornwell University extender (CUE) and named it Hannover thawing solution (HTS). In principle, egg yolk and sulfanilamide were left out. CME was also modified and named coconut milk glucose (CMG) solution. In the latter case, glucose and sodium hydrogen carbonate were added to CME and polymyxin was omitted. HTS preserves sperm motility up to 48 hours after thawing. Sperm motility for up to 72 hours after thawing in HTS at 20, 25 and 30°C was significantly better (p<0.001) than in CMG. Only at 15°C was no difference observed. However, both media produced a significantly higher percentage of motile sperm after thawing than the control samples of a glucose solution of sodium hydrogen carbonate.
Because of seasonal breeding in New Zealand, as cited by Salisbury, van Demark and Codge (1978), semen is frozen in bulk. The initially extended semen is cooled to 5°C and finally diluted to a concentration of 600 million sperm/ml without glycerol. The extended semen is then placed in dialysis tubing, about 1.5 m long and 6 mm in diameter, and suspended in an extender containing glycerol. After equilibration, the semen in the dialysis is frozen. The frozen semen is thawed at 37°C and then further diluted with Caprogen with 5 percent egg yolk and catalase (Shannon, 1968). If animals are inseminated within three to seven hours after thawing, five million total sperm per insemination are sufficient.
Fertility results with liquid and deep-frozen semen
Providing liquid semen was used within the first two days after processing, insignificant differences were found between the conception rates of cows inseminated with liquid semen and those inseminated with deep-frozen semen (Satter, Deka and Baruah, 1980; Austin, Rodricks and Rathnasabapathy, 1978). However, liquid semen seems to achieve higher conception rates if inseminations are carried out in the early oestrus phase (New Zealand Dairy Board, 1977). Using frozen semen, Schuh (1988) found that between five and ten million progressive motile sperm per insemination are needed in order to ensure optimal fertility rates. In bulls with a low fertility capacity, even more than 20 million progressive motile sperm per insemination might be required.
Satisfactory results have been claimed by different authors as regards the survival and fertility rates of bovine spermatozoa preserved in CME. In seven countries in Africa, Asia, South America and the Near East, Norman (1964) reported a conception rate of 61.5 to 39 percent when semen of three to 15 days was used for insemination.
Norman et al. (1959) conducted a trial in which 1 224 first inseminations were carried out and a non-return rate (NRR 30 to 60 days) of 70.8 resulted when seven-day-old CME RT semen was used. By using zero- to six-day-old CME RT semen, Norman et al. (1960) reported an NRR (30 to 60 days) of 79.4 in 1 125 first inseminations.
A calving rate of 65.9 percent was reported by Carrazoni and Bojanovich (1973) for Hereford cows inseminated with Brahman semen that had been preserved for up to seven days in coconut water at 15 to 20°C. In a comparison between CME and deep-frozen semen used under conditions in Uganda, inseminations with frozen semen showed better results than those with CME semen (El-Wishy, 1976). Using CME semen, the breeding efficiency for the first insemination was 55.4 percent in 1 135 inseminations, and for deep-frozen semen, 69.1 percent in 382 inseminations. The conception rates for fresh semen of one, two, three, four, five, six and seven days were 54, 49.3, 52.2, 49.2, 31 and 31.3 percent, respectively.
3 Fertility following insemination with variable numbers of sperm in Caprogen-based RT diluents
Fécondité après insémination avec un nombre variable de spermatozoïdes dans des diluants au caprogène à température ambiante
Fecundidad después de la inseminación con un número variable de espermatozoides en diluentes a base de caprógeno a temperatura ambiente
|
Diluent |
No. of sperm/insemination (millions) |
|||||
|
2.5 |
1.875 |
1.5 |
||||
|
No. of cows inseminated |
NRR (%) |
No. of cows inseminated |
NRR (%) |
No. of cows inseminated |
NRR (%) |
|
|
Caprogen (20% egg yolk) |
5614 |
63.5 |
2107 |
65.8 |
1933 |
65.2 |
|
Caprogen and catalase (20% egg yolk) |
8154 |
66.6 |
1575 |
67.2 |
1601 |
65.6 |
|
Caprogen and catalase (5% egg yolk) |
7160 |
67.4 |
1515 |
67.5 |
1699 |
68.4 |
4 Fertility rates after storage at 5°C or ambient temperature (16-20°C)
Taux de fécondité après conservation à 5°C ou à température ambiante (16-20°C)
Porcentaje de fecundidad después del almacenamiento a 5°C o a temperatura ambiente (16-20°C)
|
|
Trial 11 |
Trial 21 |
Trial 32 | |||
|
Storage time (hrs) |
5°C |
16-20°C |
5°C |
16-20°C |
5°C |
16-20°C |
|
(Percentage) | ||||||
|
6-12 |
81.1 |
79.7 |
- |
- |
66.3 |
67.1 |
|
24-36 |
74.8 |
78.6 |
84.9 |
84.7 |
68.7 |
70.6 |
|
48-60 |
79.0 |
84.1 |
80.4 |
83.0 |
- |
- |
|
72-84 |
- |
- |
81.1 |
80.3 |
- |
- |
|
(Number of inseminations) | ||||||
|
6-12 |
605 |
214 |
- |
- |
546 |
457 |
|
24-36 |
1684 |
502 |
904 |
841 |
1536 |
1595 |
|
48-60 |
205 |
69 |
850 |
938 |
- |
- |
|
72-84 |
- |
- |
334 |
317 |
- |
- |
1 NRR = 24 days.
2 NRR = 49 days.
5 Cost of different breeding models in Turkey
Coûts de différentes méthodes de reproduction en Turquie
Diferentes modelos de cría y sus costos, en Turquía
|
Model |
No. of cows served |
Calculated breeding costs (US$) |
|
Herd bull |
5-45 |
600-73 |
|
Bull shared by 10 herds |
50-450 |
60-7 |
|
Al using liquid bull semen |
2000-10000 |
19-4 |
|
Al using frozen bull semen |
2000-10000 |
40-8 |
|
LN1 facilities shared by 2 units |
|
26-5 |
1 LN = liquid nitrogen.
6 RT extenders for liquid semen and approximate storage times for optimal fertility rates
Diluants du sperme liquide à température ambiante et durée approximative de conservation pour des taux de fécondité optimaux
Diluentes para semen liquido a temperatura ambiente, y tiempo aproximado de almacenamiento para tasas de fecundidad óptimas
|
Extender |
Storage time |
Source (hrs) |
|
Caprogen |
60-84+ |
Shannon and Curson, 1984 |
|
Coconut milk extender (CME) |
120-168 |
El-Wishy, 1976; Norman et al., 1960 |
|
Cornwell University extender (CUE) |
72-120 |
Boye, 1968 |
7 Extenders for rediluting frozen semen, to be used as RT semen, and storage times for optimal fertility rates
Diluants utilisés pour rediluer le sperme congelé et permettre son utilisation à température ambiante et durée de conservation pour des taux de fécondité optimaux
Diluentes para rediluir el semen congelado y utilizarlo como semen a temperatura ambiente, y tiempo de almacenamiento para tasas de fecundidad óptimas
|
Extender for redilution |
Storage time (hrs) |
Source |
|
Hannover thawing solution (HTS) |
12-24 |
Omer, 1971 |
|
Coconut milk glucose (CMG) |
12-24 |
Omer, 1971 |
|
Caprogen |
6-36 |
Shannon, 1968 |
Fertility trials with Caprogen semen were carried out by Shannon and Curson (1984). A comparison was made between the storage temperatures of 5°C and 16 to 20°C, also considering increases in storage time (Table 4). Fertility of semen stored at 16 to 20°C was higher than that of semen stored at 5°C (p<0.06).
The mortality rate of embryos is obviously reduced by the use of fresh rather than frozen semen. For 25 heifers slaughtered a few days after insemination with fresh semen, 24 slaughtered one month after insemination with fresh semen and 24 slaughtered one month after insemination with frozen semen, the percentage of animals with a fertilized egg or an embryo was 80, 75 and 62.5, respectively. All embryos and all but one fertilized egg were viable (Boland and Gordon, 1979). Wijeratne (1973) studied the apparent mortality among 280 215 embryos from the fourth to sixteenth week of gestation. The rate of apparent mortality was higher among embryos in females inseminated with frozen semen than in those inseminated with fresh semen (24.7 versus 20.2 percent). Extracorporeal ageing of fresh semen also increased the rate of apparent embryonic mortality, but to a lesser extent than it did for frozen semen.
In ewes, fertility results with fresh semen are still superior to those of thawed semen (Salamon, 1972; Langford et al., 1979; Tervit, 1985; Trejo, 1988).Therefore, in sheep, most AI is performed with diluted liquid semen while only a few ewes are inseminated with frozen semen (Colas, 1983). Deep-frozen ram semen is not easy to use, mainly because of three factors: fertility of deep-frozen semen is about 20 percent lower than that of fresh semen; the required number of spermatozoa appears to be higher than in fresh semen; and the technique is more expensive and more complicated (Colas, 1975). In the trial of Salamon (1972), mature Merino ewes were inseminated at the second oestrus after synchronization with intravaginal sponges. Of 172 ewes inseminated with thawed semen, 52.9 percent lambed. Of 153 ewes inseminated with fresh semen, 76.5 percent lambed. Even when semen was stored for eight hours in a refrigerator, conception rates were significantly lower with refrigerated semen than fresh semen (Cordova et al., 1989).
Using rediluted bull semen in 1966, Wibling (cit. Omer, 1971) reported NRR results of 67.8 and 71.8, respectively, for 3 000 inseminations carried out with pellet semen four and six hours after thawing.
Rediluted semen (pellet semen thawed in HTS) was used by Omer (1971) in a trial of 2 547 first inseminations. With a concentration of 33 million sperm per pellet and using HTS, an NRR of 80 was achieved. This compares favourably (p<0.05) with 2 562 first inseminations which gave an NRR of 77.6 with conventional thawing solutions. Semen thawed in HTS and kept for 24 hours reached an NRR (60 to 90 days) of 70 percent in 140 first inseminations. With semen stored for 24 to 48 hours, 1 15 first inseminations resulted in a 59.1 NRR, while 35 first inseminations carried out with semen stored for 48 to 72 hours resulted in a 48.6 NRR.
Shannon (1968) rediluted frozen concentrated semen with Caprogen and 5 percent egg yolk and catalase. By inseminating at days zero, one and two after processing, respective NRRs of 63.2 (353 inseminations), 64.1 (654 inseminations) and 55 (80 inseminations) were achieved.
Benefits of liquid semen compared with deep-frozen semen
A cost-benefit analysis of different breeding models using liquid and deep-frozen semen was undertaken by Hickman and Istanbulluoglu (1984). They give comparative cow breeding costs using different breeding methods in Turkey (see Table 5).
Only in the case of the herd bull model were carrying costs used. Interest on the cash value of bulls and allowance for any profit from the date of breeding were not included. Neither was allowance made for reproductive diseases caused by natural service or for the loss of potential milk yield from the barn space occupied by the bulls.
In the case of AI models, there is no allowance for the benefit of genetic superiority over the herd bull model, nor is there any cost factor for recording systems and breed improvement programmes. The frozen model assumes the purchase of semen, with operational costs relating to storing and handling only. Neither of the AI models include transportation costs for inseminators. The authors consequently concluded that the fresh semen model is substantially less costly when the number of cows to be bred is limited.
Radhakrishna et al. (1983) estimated, to the contrary, that the effective cost for one AI with fresh semen is about three times more than it is with frozen semen. Transportation costs per insemination are more than double, and for each live calf more than triple. They therefore favoured the frozen semen system. In AI units using liquid semen, higher costs resulted primarily from the wastage of semen and reduced conception rates in the authors' experiments. The wastage of liquid semen was evident, since a minimum number of doses of semen had to be dispatched to AI units, irrespective of whether AI was carried out or not.
In the 1960s, certain advantages were discovered in using RT semen for AI in New Zealand, especially under local conditions. More than 90 percent of farmers in New Zealand endeavour to have most of their cows calve between late June and September. This means that most cows must be inseminated or mated between late September and December. In principle, one might imagine that, because of such fluctuations in demand for semen, the use of deep-frozen semen would be ideal for New Zealand conditions. Semen could be collected all year round and then used over the peak period. Initially, however, the change to a deep-frozen programme was rejected on three major grounds: conception rates obtained with deep-frozen semen were well below those obtained with liquid semen; not all semen could be deep-frozen; and the freezing and storing of semen at low temperatures would have increased costs considerably (Shannon, 1968). Work was therefore concentrated on improving the use of liquid semen.
Later, in fact, the marked seasonal pattern of requests for inseminations was also met by the development of service using semen diluted in Caprogen that contains catalase (Shannon, 1973). The diluted semen, stored at ambient temperatures, was used mostly for inseminations completed the day after initial processing. The standard dose rate was 2.5 million total sperm per insemination, in contrast to the dose rate of 25 million total sperm for deep-frozen semen. However, the use of semen processed in Caprogen meant that farmers could nominate only the breed of the sire and not the particular sire within the breed. This was because, with the use of RT semen, the bulls specifically requested might not be available on a given day (Macmillan and Curnow, 1976).
However, the use of semen rediluted at thawing with Caprogen could be considered (Shannon, 1972) a means to improve AI management in New Zealand. During the late October peak, more inseminations with semen from selected sires would be feasible with the use of deep-frozen semen rediluted at thawing with Caprogen (New Zealand Dairy Board, 1969).
By maximizing usage of the best bulls, the technique of producing RT semen treated with Caprogen also has the advantage of utilizing the genetic qualities of top sires more efficiently. In general, fresh semen allows for low dose rates (2.5 million total sperm per insemination instead of 20 million to 25 million total sperm per dose when using deep-frozen semen) and it has therefore facilitated the extensive use of selected progeny-tested sires. Demand for semen has even enabled some sires to record over 100 000 inseminations per year (Macmillan and Curnow, 1976), whereas only approximately 40 000 inseminations per year could be performed with processed deep-frozen semen.
When implemented in developing countries, technology from the developed countries must be adapted to a specific environment. In the case of AI, where self-accounting and sustainability is not evident, ways of implementation might be changed to enhance the technique's profitability and its potential in animal breeding as a whole. Developing countries produce semen mainly for their own AI use and it is not competitive with semen from developed countries. Only in rare cases is progeny testing carried out and, even then, it is on a limited basis. Consequently, most of the processed semen in developing countries is not sufficiently progeny tested. Many developing countries, moreover, are not free of contagious diseases such as foot-and-mouth disease (FMD) and rinderpest and are therefore prevented from participating in the international semen trade. There is consequently no need to store nationally produced semen, nor to make use of highly diluted semen. The often claimed wastage of genetic merit, when using fresh semen models on a small scale, is therefore not relevant to developing countries.
Thus, the use of RT semen could be most appropriate for developing countries, especially in breeding programmes using native breeds, where cow numbers are often barely large enough to carry out progeny testing and for which semen imports are of negligible breeding value. An example is the trypanosomiasis belt of West and central Africa, where this and other diseases can be fatal to all imported animals and their cross-breeds but where local trypanotolerant breeds thrive in their natural tropical environment. With a reliable public transport system, the use of RT semen could certainly be considered for such cases. Its use would lower investments and maintenance costs for the installations needed in the production and storage of deep-frozen semen. RT semen would also make AI independent from the use of liquid nitrogen. In Uganda, for example, bull semen processed as RT semen (CME) was distributed by bus to approximately 60 AI subcentres from 1960 to 1979.
Evidently some developed countries are now becoming interested in the use of RT semen because of its potentially elevated dilution rate. In Ireland, for example, following the introduction of Caprogen-RT semen, the top four bulls in Munster's 1988 proven stud averaged 34 750 first inseminations each and accounted for 72 percent of all inseminations. Almost two-thirds of the inseminations were carried out with fresh semen (Cunningham, 1989).
In the Netherlands, Caprogen-RT semen was recently distributed and used nationwide at a storage temperature of 19°C and 5°C in order to facilitate the extensive use of selected progeny-tested sires and to determine the best storage temperature.
However, it should be remembered that, even if RT semen can be stored for up to four to five days without a significant decrease in its capability of fertilization, it should be used on the same day of processing or, possibly, one day later. Despite the addition of antibiotics and antimycotics to semen diluters, several authors have suggested a daily examination of fresh or RT semen to check for contamination. In developing countries frozen semen is often thawed at the home of the technician or at the subcentre and then sometimes carried to the farmer after thawing because of the inconvenience of using a liquid nitrogen container. In such cases semen quality might well be adversely affected.
The preservation of thawed pellet semen was successfully examined by Omer (1971) and sperm motility sustained without significant loss for more than 48 hours. In New Zealand, too, the method of rediluting thawed semen with Caprogen was practiced successfully (Shannon, 1968).
To sum up the ageing factors in semen, the dead sperm enzyme aromatic-L-amino acid oxidase obviously plays a key role in the production of hydrogen peroxide and, consequently, in the deterioration of semen. Adding catalase to the extender in order to neutralize the hydrogen peroxide is a most effective form of protection against ageing effects. These results also apply to RT extenders of deep-frozen semen. Since approximately 50 percent of the sperm are killed by deep freezing, it might be possible to wash the surviving thawed sperm to separate the thawed medium as well as the dead sperm, and thus the actual resource of the dead sperm enzyme aromatic-L-amino acid oxidase, from the semen. Otherwise, freezing in a highly concentrated form could be considered.
The question raised in this article is how to apply AI most efficiently in developing countries, where the socioeconomic situation is marked by a surplus of labour, low salaries and a chronic shortage of foreign currency. Because of the latter, ways should be found to lower investments and maintenance costs which are often limiting factors to the long-term management of AI.
The wastage of semen that has a high genetic value, the desire to be independent of time and place for using such semen and the availability of LN2 at a reasonable price are leading developed countries toward the introduction of deep-frozen semen. Recent tendencies in some developed countries (Ireland, the Netherlands) have moved toward the reintroduction of RT semen because of the more extensive use of selected progeny-tested sires.
In developing countries, the validity of the above reasons does not hold, as, for example, the wastage of semen of high proven genetic merit might not normally occur. A major prerequisite for the production of deep-frozen semen seems to be the availability of LN2 at a reasonable cost. With liquid semen, a reliable, almost daily, distribution network is essential and, with such a network established, the liquid semen model can be very valuable to AI in developing countries. The use of RT semen (CME, Caprogen) would allow a storage time of at least three days. Taking into account the fact that maximum dilution rates are not required in most developing countries, a higher number of sperm per dose could help to increase fertility rates and also storage time. The increase of the latter could be brought about by less O2 tension in the semen and by the higher amount of motile sperm per dose. In this context, prophylaxis and protection against contamination of the semen will become a major issue for the preservation of RT semen.
Besides offering a far higher dilution rate and normally higher fertility rates than deep-frozen semen, liquid semen is simpler to handle. In addition, the use of liquid semen in sheep gives better results than frozen semen.
Finally, where LN2 is available at a reasonable price, a combination of both liquid and frozen semen could be envisaged. The use of rediluted semen after thawing might also be of interest to some AI management teams as it takes advantage of both systems. However, investments for both systems would be necessary in this case. Much effort should be made to produce semen with the availability of resources of the country concerned in order to contain investments and maintenance costs. AI concerns should, in one way or another, become commercial, with management itself seeking the most suitable cost-benefit solution.
Austin, A.J.S., Rodricks, I.M. & Rathnasabapathy, V. 1978. Comparative merits of liquid and frozen semen on [sic.] the conception rate in white cattle. Cheiron, 7(2): 167-173.
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