Feasibility of using hard gelatin capsules for the packaging and deep-freezing of bull semen

THERIOGENOLOGY

FEASIBILITY OF USlNC HARD GELATIN PACKAGING AND DEEP-FREEZING A.J.

1 Bombay 2

Thanawala, Department Veterinary

’ S.A. Sonawanef of Gynecology College, Parel,

CAPSULES FOR THE OF BULL SEMEN and C.R.

Sane’

and Obstetrics, Bombay 400 012,

India.

SciTech Centre, 131, Kandivli Industrial Estate, Kandivli (West), Bombay 400 067, India.

Received

for publication Accepted

: ~pri2 :

IS, 2987

December

13,

1987

ABSTRACT Various packaging systems have been used for deep freezing of semen. In this study, feasibility of using hard gelatin capsules was established. Of the four types of capsules developed and tested, polymer-treated capsules used subsewere found to be suitable for the purpose, and were therefore quently. French medium (0.5 ml) straws were used for controt. Five semen samples from each of 12 bulls were processed and included for study. Parameters studied after thawing Semen was frozen by fast-freezing. of semen were comparable for the two methods. Upon analysis, the percentages of progressive motile spermatozoa, live spermatozoa and morphologifor semen frozen in hard gelatin cally abnormal spermatozoa obtained The capsules and French medium straws were found to be nonsignificant. percentage of intact acrosomes was found to be significantly higher in straws as compared to semen in (P< 0.05) for semen frozen - thawed capsules. Key words

: capsule,

straw,

deep freezing,

semen,

cow-bull

INTRODUCTION Deep--freezing of semen has brought about a phenomenal change in the field application of artificial l’nsemination for intensive testing, selection Frozen semen has made and use of proven sires for breed improvement. possible the long distance transport and the long-term storage of semen of valuable sires. The use of frozen semen has brought sweeping changes in the laboratory processing, packaging, storage and shipment of semen.

Acknowledgments This study was supported by a research grant from SciTech Centre, Bombay, India. Authors are grateful to h\r. Ajit Singh for his encouragcment and Dr. A.V. Patwardhan for research co-ordination. Thanks to Dr. S.R. Lele for help in developing capsules and Dr. L.K. Sharma for excellent technical assistance.

APRIL 1988 VOL. 29 NO. 4

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THERIOGENOLOGY

Initially, semen was frozen in glass ampules or vials. Later, a method of freezing semen involved extending semen at lower dilution rates and freezing it as pellets, virtually eliminating extender volume and packaging containers. However, no reliable method of direct insemination of pellets was available. This fact, together with the lack of positive identification for semen so processed, discouraged the widespread use of pelleted semen in the field. A Danish researcher, Sorenson, first developed the idea of packaging and freezing extended semen in plastic straws in 1940. Since then, a number of workers have recognized the advantage of such a system for practical use by the A-1. Organizations. Reports of higher conception rates, greater spermatozoa1 utilization and greater storage efficiency are the salient features of the straw method. A French researcher, Cassou, refined the technique. Little storage space is required and good sperm survival is reported .with the Cassou straw. Existing methods of freezing and packaging are constantly reviewed and new methods are being developed. The new methods aim at reducing cost and storage space along with improving efficacy of processing and packaging while maintaining good sperm survival for a high conception rate. Cur study was undertaken I) to develop suitable capsules to hold semen; 2) to develop a new method for packaging and freezing semen in hard gelatin capsules; 3) to compare French medium (0.5 ml) straws and the newly developed hard gelatin (0.5 ml) capsules. MATERIALS

AND

nlETHODS

This study was carried out at the Department of Gynecology and Obstetrics, Bombay Veterinary College, Bombay, and Development Corporation of Konkan Ltd., Unit No. 16, Aarey hlilk Colony, Coregaon, Bombay, for a period of six months from June 1985 to November 1985. Three bulls each of Holstein-Friesian and Jersey breeds (purebred) and Holstein-Friesian x Gir and Jersey x Cir breeds (50:50) were selected. All 12 bulls were clinically healthy and were showing good sexual behavicur. Semen was collected from them at weekly intervals and was found to be suitable for freezing. The extender was and 6 ml of glycerol.

prepared

The four different types gelatin capsules, wax-treated

with

74

of capsules,a hard gelatin

ml

tris

buffer,

20

1111 egg

yolk

were used: plain untreated hard capsules, liquid paraffin-treated

hard gelatin capsules, and polymer-treated hard gelatin capsules. A trial using 20 capsules of each type was undertaken to determine the type of capsule suitable for the purpose of deep freezing. An aluminiurn capsuleholder was specially designed and fabricated (Figure No. I). The holder was provided with holes to ensure maximum contact of the capsules with nitrogen vapours during freezing. Each holder could hold upto 18 capsules at a time (Figure No. 2).

aAssociated

922

Capsules

Group

and SciTech

Centre,

Borrlbay,

India.

APRIL 1988 VOL. 29 NO. 4

THERIOGENOLOGY

Figure

Figure

I.

2.

The Aluminium holes on both

Bottom Top

capsute-holder with through holes. the ends are for the purpose of lifting

: Capsules held vertically : Capsule-holder inserted

APRIL 1988 VOL. 29 NO. 4

on holder. partially into

The

small

the holder.

the goblet.

923

I-HERIOGENOLOGY

Semen collections were obtained frotn bulls by rncans of an artificial immediately vagina at weekly intervals in the early hours of morning. after collection, the semen in the graduated tube was placed in a water Semen sample with optimum activity (+++) and initial bath at + 30°C. The motility (+3) was processed further immediately after collection. samples with initial motility above +3 and sperm concerntration above 800 million/ml were used for extension. Depending on total sperm concen tration estimate, the semen was further extended to give a final concentration of 40 million spermatozoa/O.5 ml of semen. selected from each of the 12 bulls A total of five ejaculates were for extension, filling, freezing and further evaluation. Thus 60 semen samples were included for the study. After initial evaluation, the extended semen was cooled slowly to +5”C and equilibrated at this temperature for 6 h. Empty capsules were placed into position on the holder and cooled to +5”C. A quantity of 0.5 ml of equilibrated semen was transferred into each of The capsules were then the capsules with the help of a sterile pipette. closed with the help of tight fitting caps. The straws were filled with the help of semiautomatic filling machines. Freezing

of Straws

and Capsules

The equilibrated straws were placed on racks along with hard gelatin capsules before carrying out vapour freezing. The equilibrated straws and capsules were vapour frozen in the LR-320 (Union Carbide) container which was prepared with a proper amount of liquid nitrogen upto the grill 20 min before freezing. These straws and gelatin capsules were frozen -4 cm above level for 10 mtn to achieve a temperature of -140°C. Thus, the average freezing rate was - 14.5”C/min. The straws and capsules on each rack were collected and quickly immersed in liquid nitrogen and transferred to smaller storage containers. Analysis

of Extended

and Frozen-Thawed

Semen

Varrous parameters were studied for the extended sernen and the frozenthawed semen in the drfferent packaging systems. Thawing was carried out in a water bath at +37”C for 60 sec. The percentage of progressive Combined motile spermatozoa was estimated by using a haemocytometer. eosin-nigrosin stain (I) was used to calculate the percentage of live and abnormal spermatozoa according to methods described elsewhere (2,3). The Giemsa’s rnethod was used to evaluate the percentage of intact acrosomes. RESULTS Four different efficacy in holding semen quality upon of containers appear

924

types of hard gelatin capsules were the semen, facilitating deep freezing thawing. The properties of each of below (Table I).

tested for their and maintaining the various types

APRIL 1988 VOL. 29 NO. 4

THERIOGENOLOGY

Table

1.

The

efficacy

the

semen,

quality

upon

of the facilitatmg

Plain

capsules in maintainrng

of

capsules

that maintained retained semen After freezingthawing

0 (20)

Wax-treated

12 (20)

0 (12)

Liquid treated

8 (20)

0 (8)

paraffin-

Polymer-treated in Parenthesis

20 (20)

18 (19)

19 (20)

indicate

the

number

All 20 plain untreated capsules lost soft and leaked their contents. Hence for the purpose of further processing.

of

18 (18)

capsules

their they

tested.

normal consistancy, were found to be

Of wax-treated capsules, 12 out of 20 were found semen well. After deep freezing, however, they lost their leaking soon after being taken out of the liquid nitrogen. Of to hold leaking

holdrng semen

Number of capsules showing optimum sperm motility

shape, consistancy and After Before freezing freezing

untreated

Figures

hard gelatin freezing and

thawing

Number Types of hard gelatin capsules :

different deep

the liquid paraffin-treated the liquid semen. Upon and/or deformed.

Polymer-treated hard when filled with semen, the capsules, except one, Upon thawing, 18 out of sperm motility.

deep

capsules, freezing,

only all

8

out

8 were

became unsuitable

to hold the liquid shape and started

of

20

found

were to be

found either

gelatin capsules did not lose their consistancy All and no leakage of contents was observed. withstood deep freezing and retained their shape. 19 capsules remained intact and showed optimum

Thus the polymer-treated hard gelatin capsules were found to be the However, for further trials, the process best among the four capsules tested. of polymer-treatment was improved to ensure uniform coating of the polyrnrr Subsequently, throughout the inner and outer surfaces of the capsules. there was no leakage of semen after thawing.

APRIL 1988 VOL. 29 NO. 4

925

rI-IERIOGENOLOGY

Table

2.

The mean abnormality and after treated

valuesa of percentages and intact acrosomes freezing-thawing for

hard

gelatin

capsules

and

for progressive motility, of spermatozoa before the

semen

French

packed

medium

Progressive percentage

Hard gelatin capsule

68.81 f 1.13

47.63 r 1.28

47.72 ? 1.40

percentage

83.26 * 1.13

52.56 f 1.26

50.46 + 1.26

Morphologically abnormal percentage

9.75 r 0.86

15.30 * 1.07

14.80 + 0.98

Percentage acrosome

intact

84.46 f 0.51

57.73 f 0.90

f

aThe

Mean

f SEM

Table

3.

Live

motile

freezing-thawing

French medium straw

freezing

polymer-

straws

After Before

Spermatozoa1 characters

in

livability, freezing

obtained

for

of

Between mean

characters motile

Morphologically percentage

aAll b

NS

the

intact

values

for progressive motility, acrosomes of spermatozoa

\Vithin mean

method square

Variance component ‘F’ Value

22

were

0.29

16.31

10.54

3.25

3.23

I.18

7.9

1.5

43.57

5.87

angle-transformed

before

0.01

NSb

NS NS

1.41*

analysis.

= Non-significant.

*Significant

926

abnormal

percentage

method square

processed.

:

percentage

Percentage acrosome

samples

I

freedom

Spermatozoa1 Progressive per ten tage Live

60 semen

Analysis of variancea of percentages livability, abnormality and intact for the two methods of packaging

Sources of variation

Degrees

the

53.5 1.00

(PtO.05).

APRIL 1988 VOL. 29 NO. 4

THERIOGENOLOGY

DISCUSSION The use demonstrated

of gelatin capsules for packaging (4). A technique was developed

by

semen which

for A.I. has semen pellets

been are

frozen in gelatin capsules which could be labelled and used for insemination either thawed or frozen (5,6). A method of freezing bull semen in gelatin capsules was also found (7). As compared to the earlier used pellets, the use of gelatin capsules enables each dose of semen to be labelled and eliminates the necessity of using dry ice ,and it protects the semen against contamination. The semen dispensed in the form of capsules gives a better conception rate than pelleted semen (8). the rnodified polymer-treated Of the various types of capsules tested, hard gelatin capsules were found ideal for packaging and deep freezing It was observed that semen. However, further improvement is needed. while the caps of the capsules were tight-fitting, they were not airtight. the air space inside the capsule got After immersion into liquid nitrogen, The semen was thus exposed to contamination. filled with liquid nitrogen. Also. at This can be avoided by developing airtight seals for the cap. present, the capsule-holder accommodates just eighteen capsules per goblet. A more extensive arrangement is desirable to make it economical and commercially viable. The French medium (0.5 ml) straw was chosen as a control. method has been found to be better than the capsule method storage and optimum motility upon thawing (9, IO). The progressive from large to medium (0.5 ml) straws requires (ll,lZ). Thus the French medium (0.5 ml) straw method of packaging and freezing semen.

The straw for semen reductiori

less storage space and time has been taken as a standard

Various types of materials have been tried for packaging semen. Class than plastic containers to prcservc contamers were found to be better motility of sperms (13). However, tube polythcnr was cc-onornirat and convenient (14). Gelatin is a suitable material for packaging semen (5,7). For this study, polymer-treated hard gelatin capsules were developed. The French medlurn (0.5 ml) straws were kept as a control. The percentage of progressive motile spermatozoa after thawing \\‘a~ 47.72 + 1.4 for tlard gelatin capsules. This was slightly higher than the value of 47.63 k 1.2s (Table 2) obtained for the French mediurn straws. The difference was nonsignificant (Table 3). It can be said that the progrcssivc motile spermatozoa are preserved as effectively in hard gelatin capsules as in French medium straws. The percentage of live spermatozoa after thawing wa< 50.46 + 1.26 for hard gelatin capsules for 52.56 t 1.26 for French medlurn Straws (Table 2). The difference was found to bc nonsignlficant (Table 3). Thus, hard gelatin capsules effectively preserve the viability of spcrrn.

APRIL 1988 VOL. 29 NO. 4

927

II-IERIOGENOLOGY

The

percentage

t 0.98 for hard of 15.30 + 1.07 the difference capsules are medium

and

of gelatin (Table

morpohologically

abnormal

spermatozoa

was

14.80

the

value

This was slightly lower than in French medium straws.

capsules. 2) obtained

was nonsignificant (Table as effective in preserving

3). the

Polymer-treated live spermatozoa

However,

hard as

gelatin French

straws.

The percentage 57.73 +_ 0.90

intact acrosomes for French medium

was 53.5 + 1.0 straws (Table

for hard 2); this

gelatin capsule difference was

found to be statistically significant (Table 3). It can be said that the polymertreated hard gelatin capsufe is not as efficient as the French medium straw in maintaining the percentage intact acrosomes. The reason could be the slower thawing of capsules due to their greater diameter. it was found that polymer-treated hard gelatin capsules are Thus, as efficient as French medium straws in maintaining the quality of semen after freezing-thawing for all parameters, except the percentage of intact The polymer-treated capsules developed in this study were acrosomes. found to be suitable to withstand the rigours of deep-freezing and thawing. a reliable method of insemination remains to be developed, and However, the conception rates need to be determined for the method to become practically viable under field conditions.

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3.

J.L.

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( 1945).

4.

Davis, H.P., Underbjerg, C.K.L. and Trimberger, C.W. An improved method for artificial insemination of the bovine by vaginal deposition of the semen. Am. Sot. Anim. Prod. Proc. z22 l-223 (1940).

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Merkt, pellets

6.

K.F. and Lorrmann, Merkt, H., Weltze, W. Semen pellets sealed in a capsule, a means of improving the pellet method in the deep frozen storage of bull semen. Anim. Breed. 36:3598 Abstr. (1967).

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Pilch, J. The freezing 3251 Abstr. (1971).

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K.F. and H., Weitze, in capsules on a practical

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Burnkhorst, F. Cold scale. Anim. Breed.

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Anim.

Breed.

with (1966).

39:

APRIL 1988 VOL. 29 NO. 4

THERIOGENOLOGY

8.

Pavlenko, hl.P., Kuznetsov, G.T., Tsybuinik, V.T. and Ostashko, F.I., The technology of freezing bull semen in encapsulated M ischenko, N. Tekhnologiya Kriokonservatii spermy bykov v oblitsovannykh pellets. granulakh. Zhivotnovodstvo 11:54-56 ! 1978).

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Cassou, R. of freezing.

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Linares, and in ( 1969).

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Cassou, R. Miniaturization Paris, p. 163 (1968).

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Dimitripoulos, E. Trials for improving the composition of bull diluents and the freezing technique to obtain better fertility. Med. Vet. u:184-201 (1969).

13.

Musgrave, deep-frozen

14.

Roy, D.J. A new field technique semen in Tupol. Ind. Vet. J. 51:249

APRIL

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929