Survival of Bull and Ram Spermatozoa in Preparations from Skimmilk

Survival of Bull and Ram Spermatozoa in Preparations from Skimmilk R. ¢. JONES Department of Animal Science, Corne]l University, Ithaca, New York Abstract

Media containing the nondialyzable components of skimmilk were prepared in dialysis sacks by placing the sacks filled with skimmilk in buffered saline and storing at 5 C for 48 hours. Similarly, media containing the dialyzable cmnponents of milk were prepared by placing dialysis sacks of buffered saline in skimmilk. When these preparations (alone and in combination) were mixed 1:1 with a solution of 185 mM lactose and electrolytes, bull and ram spermatozoa survived at 37 C better in the nondialyzable than in the dialyzable preparations, and either of these preparations was better than a 1:1 mixture of both. t t e a t i n g the dialyzable preparation or mixtures of both preparations before use increased survival rates, but heating rendered the lactose solution harmful to bull and ram spermatozoa. When the concentration of sodium chloride was varied in buffered saline diluents containing heated dialyzable and unheated and heated nondialyzable milk preparations, it was found that media containing the nondialyzable preparations had a higher tonicity (measured by the survival of bull spermatozoa) than the dialyzable preparations. Heating the nondialyzable preparations improved the survival of spermatozoa. Both preparations, either heated or unheated, were lyophilized and used in a factoriM experiment at concentrations of 0.5 and 1.0% w/v in media of relative tonicities 0.8 and 1.0. Scores of survival of spermatozoa made after incubation at 30 C for eight hours were lowered by increasing the concentration of the lyophilized preparations and increasing the tonicity of the diluents, ttowever, mean scores of per cent motile spermatozoa in samples from media containing unheated dialyzable and nondialyzable preparations were 25.4 and 24.6, respectively; for media containing preparations heated before lyophilization, the means were 18.8 and 33.8.

Received for publication Jaauary 2, 1968.

Thacker and Ahnquist (26) found that fresh cow milk is toxic to bull spermatozoa and later (27) defined the toxic factor as being inactivated by heat at 92 C, nondialyzable, and present in the sermn protein fraction of milk. Flipse et al. (11) confirmed opinions that lactenin, the antistreptococcal factor in milk (16), is toxic to spermatozoa and (15) presented reasons for their conclusion that heating milk activated sulfhydryl groups in fl-lactoglobulin which rendered laetenin nontoxic to spermatozoa. Saacke et al. (25), working with bull semen, found that owing to variations between batches of skimmilk, different batches may require different degrees of heat treatment. Blaekshaw (2) found that for ram semen it is sufficient to heat pasteurized milk at 85 C for five minutes. However, heating fresh milk at 92 C for ten minutes is now accepted as best for bull (21) and ram semen (20). The majority of reports on the fertility of ram and bull semen after storage show that diluents prepared from milk compare favorably with conventional yolk diluents (9, 21), and reasons for a number of contradictory reports (7, 12-14, 24) are not fully understood. F o r this reason the following experiments determine the suitability of a number of preparations from bovine skimmilk for storing bull and ram spermatozoa. Experimental Procedures

Ram semen was collected following electrical stimulation (1), and bull semen with an artificial vagina, and only samples containing a high proportion of spermatozoa with good activity were diluted within 45 minutes of collection for use in the experiments. Bovine skimmilk was prepared by centrifugation of recently collected milk at ],900 g at 3-5 C for 20 minutes to form a supernatant plug of milk fat from which skimmilk was easily separated. Media containing the nondialyzable components of milk were prepared by pla~ing five dialysis sacks, 1 each containing 10 ml of skimmflk, in a cylinder of one liter of 1 ¥isking was used for the studies of ram semen, a~d Cellophane (Will Scientific, Inc., USA) for the studies of bull semen. 1288

SURVIVAL OF SPERMATOZOA buffered sodium chloride ( E x p e r i m e n t s 1, 2, and 3) or water ( E x p e r i m e n t 2) and storing at 5 C f o r 48 hours. A f t e r the first 24 hours of storage, the buffered saline or water was replaced with a fresh preparation. A similar procedure, reverse dialysis, was used to p r e p a r e media containing the dialyzable components of milk. Thus, dialysis sacks containing buffered saline were placed in two changes of skimmilk. H e a t i n g of these p r e p a r a t i o n s refers to heating at 92 C f o r ten minutes immediately a f t e r dialysis. Solutions were p r e p a r e d f r o m A.R. grade chemicals assuming 154 m M sodium chloride, 100 mM phosphate buffer ( p H - - - - 7 ) , and 308 m~I sugar solutions are isotonic (relative tonicity = 1.0) with ram and bull spermatozoa (3, 19). The composition of the isotonic buffered sodium chloride solution was: 142 m ~ sodium chloride, 4 mM potassium chloride, 2.5 mM monobasic sodium phosphate, and 2.5 relY[ dibasic sodium phosphate. The lactose solution used in E x p e r i m e n t 1 had the same concentration of potassium and phosphates as the b u f fered sodium chloride solution, 185 mM lactose, and 54 m M sodium chloride. All of media used for extending semen contained 11.4 mlV[ fructose, antibiotics (500 I U penicillin per milliliter and 500 ~g streptomycin p e r milliliter), and the concentrations of potassium and phosphates stated above. Relative tonieity was varied as an experimental factor in E x p e r i m e n t s 2 and 3 by varying the concentration of sodium chloride. Semen was diluted 20- to 25-fold to avoid the harmful effects of high dilution rates. A t hourly intervals during incubation, subsamples were examined as a thin film between a slide and coverslip on a microscope w a r m stage at 38 C, using a magnification of 3 2 0 × , and scores of the rate of progressive motility [i.e., motility score, range 0-4 and increments of 0.5; (8)] and percentage of motile spermatozoa were made. The scores of motility, doubled to remove fractions, were used as unit observation in the analysis of variance (5), and scores of percentage motile spermatozoa were transformed to angles (6) for analysis. Generally, the orthogonal polynomial coefficients tabulated in F i s h e r and Yates (10) were used to partition levels of a factor. However, to test some of the hypotheses examined, other sets of orthogonal coefficients have been used. As an example of these, the set used to partition F a c t o r B in E x p e r i m e n t 1 is shown in Table 2. Those used in E x p e r i m e n t 2 were computed in a similar manner and are described in the source-ofvariation column of Table 5. The analyses of

1289

TABLE 1 Experiment 1. Mean scores of survival of ran] and bull spermatozoa after incubation at 37 C for 2 hours. A 2 × 7 factorial experiment replicated with four ejaculates from different males.) Ram semen Rate of motility

Factors and levels

Bull semen

% Motile

Rate of motility

% Motile

48.8

2.32

46.8

34.8

1.98

41.4

33.1

1.27

25.6

60.0

2.71

56.3

16.3

0.71

5.0

1.94

31.9

2.08

52.5

2.81

57.5

2.41

55.0

2.25

40.0

3.33

63.1

3.13

53.8

2.65

51.3

Preparations of milk mixed 1 : 1 with: Unheated lactose solution 2.61 Heated lactose solution 2.09 Treatment of milk 10rep before mixing 1:1 with lactose solution Not heated 1. Dialyzable preparation 1.75 2. Nondialyzable preparation 3.00 3. Prep. 1 and 2 mixed 1:1 1.56 Heated 4. Dialyzable preparation 5. •ondialyzable preparation 6. Prep. 1 and 2 mixed 1 : 1 and heated 7. Prep. 4 and 5 mixed 1:1

variance are shown in summary f o r m and the error mean square is composed of interactions higher than first order. Results

E x p e r i m e n t 1 was designed

(a 2 X 7 X 4

TABLE 2. Orthogonal coefficients used, for between milk preparations contrasts, in the analyses of variance of responses in Experiment 1. Treatment no.

Contrast no. ]

2

3

4

5

6

1 2 3 4 5

-1 1 0 -1 1

-1 -1 0 1 1

1 -1 0 -1 1

0 0 -2 0 0

0 0 0 0 0

-3 -3 4 -3 -3

6

0

0

0

1

7

0

0

0

1

-1

4

I

4

J. DAIRYSCIEI~CEVOL. 51, NO. 8

JONES

1290 TABLE 3.

Experiment 1.

Source of variation

Summary of the analyses of variance.

Degrees of freedom

Heating lactose solution (A) Milk preparations (B) Used alone 1 Dia]yzable vs. nondialyzablo 2 Not heated vs. heated 3 Interaction of 1 and 2 Mixed 1:1 4 Not heated vs. heated 5 tteated before vs. after mixing 6 Used alone vs. mixed 1 : 1 Ejaculates (C) Interactions A×B AX1 Remainder A×C B×C Residual

Ram semen

Bull semen

Variance ratios

Variance ratios

Rate of motility

1

8.25 ~

1

19.85 ~ *

Rate of motility

% Motile 14.59 ~

32.58 " H

8.48 ~

12.9 o ~

0.34 5.99 ~

8.02 ~ 9.52 ~

50.90 ~*~ 2.20

134.01 *~* 2.90

1 1

0.0 0.68

0.10 0.11

1 1

14.84 ~ 6.73 ~

27.46 ~*~ 3.44

1

0.13

4.53 ~

3

21.41 H~

1 5 3 18 18

4.40 0.53 1.26 1.70 1.82 ~

% Motile

2.80

0.89

3.09

11.70 ~

20.06 ~r~

4.41 ~

1.79

5.48 ~ 1.44 0.84 1.70 99.45"

3.05 0.63 3.11 0.71 2.30 a

0.46 1.56 3.06 0.94 70.0 ~

"Variance. * P < 0.05. *~ P < 0.01. *** P < O.O01.

factorial design) to determine i f the toxic fact o r ( s ) in milk are dialyzable. A s h e a t i n g was used to remove the toxic f a c t o r ( s ) , the effect of h e a t on lactose, a m a j o r constituent o f milk, was also tested. Table 1 shows how u n h e a t e d and heated, dialyzable and nondialyzable p r e p a r a t i o n s f r o m skimmilk were used alone and in combination. To decrease the toxicity of these p r e p a r a t i o n s , so t h a t s p e r m a t o z o a would n o t be r e n d e r e d immotile immediately u p o n dilution, t h e y were mixed ] :1 with u n h e a t e d or heated lactose solution before use. The analyses of variance ( s u m m a r i z e d in Table 3) show: F o r both scores o f survival o f r a m s p e r m a t o z o a a) h e a t i n g the lactose solution was disadvantageous, b) the dialyzable p r e p a r a tion of milk was more toxic t h a n the n o n d i a lyzable p r e p a r a t i o n , a n d c) it was beneficial to h e a t m i x t u r e s of the milk p r e p a r a t i o n s bef o r e use. F u r t h e r , scores of motility show t h a t d) it was b e t t e r to heat the p r e p a r a t i o n s o f milk b e f o r e r a t h e r t h a n a f t e r mixing, a n d over-all e) more s p e r m a t o z o a survived incubation~ i n the milk p r e p a r a t i o n s used alone r a t h e r t h a n in combination. The A × (1) c o m p o n e n t o f t h e i n t e r a c t i o n A × B shows that, f o r media J . DAIRY SOIE:~CE ~V-o/~. 51, N O . 8

TABLE 4. Experiment 2. Moan scores of survival of bull spermatozoa a f t e r incubation at 37 C for two hours. Factors and levels Preparations from skimmilk Milk preparation

Heat treatment

Motility

% Motile

NaC1

Heated

1.92

28.8

hTaC1

--

1.63

19.3

NaC1

Heated

2.54

36.7

Water

--

1.63

25.0

Water

Heated

2.46

37.1

Theoretical tonieity of final diluent 0.6 2.43 0.9 3.23 1.2 1.83 1.5 0.63

28.0 54.3 30.3 4.8

Ejaculates 1 2 3

13.8 37.6 36.8

Dialyzable Nondialyzable Nondia]yzable Nondialyzable Nondialyzable

Dia]yzed against

1.20 2.45 2.45

SURVIVAL

OF SPERMATOZOA

TABLE 5. Experimeht 2. Summary of the analyses of variance. Degrees

Source of variation

of freedom

Yariance ratios Motility

% Motile

0.88

0.07

Preparations of skimmilk A 1 Dialyzable vs. nondialyzable 1 2 Heating nondialyzable prep. 1 3 Dialysis of nondialyzable prep. against buffered NaC1 vs. water 1 4 Interaction of 2 and 3 1

39.76***

Theoretical tonleity B 1 Mean of 0.6, 0.9, and 1.2 vs. 1.5 1 2 0.6 vs. 1.2 1 3 Mean of 0.9 vs. mean of 0.6 and 1.2 1

169.64"** 11.68"*

170.15"** 1.16

52.36***

69.60***

44.12"**

0.09

1.17

0.09

1.37

Ejaculates C

2

45.08***

50.48***

Interactions AXB 1X2 3X2 Remainder AXC BXC Residual

1 1 10 8 6 24

30.33 ~** 6.49 ~ 1.01 2.59* 3.61" 0.924

24.86*** 2.97 0.89 2.59* 3.47* 39.4~

Variance. * P < 0.05. ** IP < 0.01. *** :P < 0.001. containing dialyzable preparations of milk, a greater percentage of spermatozoa survived incubation in the presence of unheated than heated lactose (i.e., mean score of 30.6 vs. 21.9% motile spermatozoa), whereas for media containing nondialyzable preparations of milk the mean scores were much the same (i.e., 58.8 vs. 58.8). The effects b), c), and e) just described for ram spermatozoa were also observed in the study of bull spermatozoa. Indeed, separate analyses of scores of survival of bull spermatozoa made after incubation for three hours (scores for Level 3 of Factor B were zero, so were excluded from these analyses) showed that the effect on bull spermatozoa of heating the lactose solution was the same as described in a). Thus, for media containing unheated and heated lactose, the mean scores of motility were respectively, 2.31 and 2.00 (F(tII~) ----5.34, P < 0.05) and mean scores of per cent motile were

1291

49.2 and 35.2 (F(l/~5) ----22.60, P < 0.001). Contrast 3) of Main Effect B (Table 3) shows that bull spermatozoa survived better in the presence of heated than unheated dialyzable milk (both scores o f survival), but heating the nondialyzable preparation slightly lowered the mean motility score and had no effect on the mean percentage of motile spermatozoa, The p}t values of dialyzable and nondialyzable milk prepared by dialysis against buffered sodium chloride were 6.69 and 6.91, respectively; therefore, it is unlikely that differences in toxicity of the preparations can be attributed to differences in pH. However, as they may differ in tonicity, this factor was tested in Experiment 2. Table 4 shows the factors and levels in this 5 × 4 × 3 factorial experiment in which the skimmilk preparations were mixed 1:1 with buffered sodium chloride. Relative tonicities were calculated assuming that the preparations from milk made by dialysis against buffered sodium chloride had relative tonicities of 1.0 and those prepared by dialysis against water had relative tonicities of zero. This assumption was justified by measurements (Advanced Osmometer, Advanced Instruments, Inc.) of osmolarity. Thus, the osmolaritics (osmolcs) of water and the nondialyzable milk prepared by dialysis against water were much the same (i.e., 0.003 vs. 0.008). Also, the osmolarities of 154 mM NaC1, and nondialyzable and dialyzable milk prepared by dialysis against 154 mM NaC1 were much the same (i.e., 0.285, 0.284, and 0.279, respectively). The analyses of variance (summarized in Table 5) show that spermatozoa survived better in heated than unheated nondialyzable milk preparations. On the average, survival was best in media of 0.9 relative tonicity (contrast 2) of the Main Effect B for scores of motility is not statistically significant when tested against B × C interaction). The 1) × 2) contrast of the interaction A × B shows that the tonicities of the dialyzable and nondialyzable milk preparations differ. F o r example, the mean score of percentage of motile spermatozoa in media of relative tonicities 0.6 and 1.2 a n d containing preparations of dialyzable milk were 8.3 and 45.0, respectively, and the corresponding means for media containing preparations of nondialyzable milk were 32.9 and 26.7. Further, the 3) × 2) contrast of the interaction A × B shows that in media with the same theoretical tonicity, the mean motility score for samples containing nondialyzable milk prepared by dialysis against buffered sodium chloride differs from the mean for samples con~. DAIRY SCIENCE VOL. 51, NO. 8

1292

ZONES

taining nondialyzable milk p r e p a r e d by dialysis against water. Thus, mean scores for the f o r m e r p r e p a r a t i o n in media of relative tonicities 0.6 and 1.2 are 2.50 and 1.92, respectively, and for the latter p r e p a r a t i o n are 2.92 and 1.33. As the dialyzable and nondialyzable p r e p a r a tions used in the previous experiments gave different concentrations of solids when lyophilized, this could account f o r differences in toxicity. F o r this reason the p r e p a r a t i o n s were lyophilized and used at equal concentrations in E x p e r i m e n t 3 (Table 6). The percentage of dry matter recovered f r o m the dialyzable and nondialyzable preparations was 6.2 and 4.1% w / v , respectively. Low concentrations of the milk preparations were used so that they would have little effect on the tonicity of the diluents. However, to test the importance of possible confounding between milk preparations and tonicity, the latter was varied as an experimental factor. Relative tonicities of 1.0 and 0.8 were used to compare what was accepted as isotonic with spermatozoa with what appeared to maintain the best survival in E x periment 2, when the results of this experiment were examined graphically. F u r t h e r , so that spermatozoa would in some samples not become immotile as rapidly as in E x p e r i m e n t s 1 and 2, the samples were stored at 30 C. The analyses of variance, summarized in Table 7, show that over-all, increasing the conTARLE 6. Experiment 3. Mean scores of survival of bull spermatozoa after incubation for eight hours at 30 C. Factors and levels

Motility

% Motile

Treatment of milk preparation before lyophilization Unheated Heated

1.58 1.81

25.0 26.3

Lyophilized preparation Dialyzable Nondialyzable

1.77 1.63

22.1 29.2

Concentration of lyophilized preparation 0.5% w/v 1.0% w/v

2.19 1.21

35.4 15.9

Degrees

Source of variation Heating milk preparations A Lyophilized preparation B Concentration of lyophilized preparation C Relative tonieity D Ejaculates E Interactions A X B A X C B X D Pooled remaining treatment X treatment interactions Pooled treatment >( ejaculate interactions Residual

of freedom

Motility

% Motile

1

3.08

0.60

1

1.25

1.78

1 1 2

56.20 ~ 66.17 ~ 33.07 * ~

44.54 ~ * 26.30 ~ 13.55 ~ *

1 1 1

2.06 4.30 ~ 4.30 ~

7.70 ~* 2.28 9.34 ~

3

0.29

0.89

8 27

1.22 0.82 ~

0.65 67.5"

* Variance ~ P ~ 0.05 . * ~ P < 0.01 . * ~ P < 0.001. centration of the lyophilized preparations or increasing the tonicity of the media decreased survival of spermatozoa. However, a number of interactions between factors were statistically significant. Thus, the interaction A × B shows that for spermatozoa incubated in media containing unheated dialyzable or nondialyzable milk preparations mean scores of per cent motile spermatozoa were much the same (i.e., 25.4 vs. 24.6), but these means were respectively lower and higher when the preparations were heated (i.e., 18.8 vs. 33.8). The A × C interaction shows that the effect of increasing the concentration of milk p r e p a r a t i o n s caused a greater lowering of motility scores when the preparations were unheated (i.e., mean of 2.21 vs. 0.96) than when they were heated (mean of 2.17 vs. 1.66). F i g u r e 1 illustrates the interaction ]3 × D for scores of p e r cent motile spermatozoa. Discussion

l~elative tonicity of media 0.8 1.0

2.28 1.17

34.0 17.4

Ejaculates 1 2 3

0.97 1.91 2.22

16.0 25.7 35.3

J. DAIRY SCIENCE VOL. 51, NO. 8

TABLE 7. Experiment 3. Summary of the analyses of variance.

Generally, it has been accepted that heating at 92 C for ten minutes, as used in these studies, is not harmful to lactose in solution (22), although if milk protein is present some combination of lactose and protein m a y occur (23). The detrimental effect of heat on lactose, as observed in E x p e r i m e n t 1, m a y explain some

SURVIVAL

50

-- • -

DIALYZABLE NON

ILl ..I u

OF SPERMATOZOA

DIALYZABLE

40

O o


30

Z ILl ~J eu, a.

Z ILl

20

10

I 0.8

RELATIVE

I i.0

TONICITY

FIG. 1. Effects of relative tonicity on percentage of motile spermatozoa after incubation for eight hours at 30 C in medium containing preparations of dialyzable and nondialyzable skimmilk. adverse reports of survival (12, 27) and fertility (7, 13, 14, 24) of bull spermatozoa diluted in preparations from milk. Indeed, some of the adverse effects of heating milk, attributed by Saacke et al. (25) as due to variations in the resistance of proteins to denaturation, may be a measure of the effect of heat on lactose. Further, the reason spermatozoa did not survive as well in media containing a preparation of dialyzable milk when it was heated rather than unheated [ (27) and Experiment 3] may be attributed to the effect of heat on lactose. The finding in these studies that both the dialyzable and nondialyzable components of milk are toxic to spermatozoa disagrees with the conclusions of previous workers (15, 27). This disagreement may be due to the use of dialysis membranes from different sources, a difference in storage temperature of semen (i.e., 30-37 C in these studies vs. 4-5 C in previous studies) or confounding by the earlier authors (15, 27) of the effects of diluent tonicity with the comparison of milk preparations. Also, the

1293

different results may be due to a difference in the composition of the media used for mixing with the milk preparation before semen dilution "[i.e., the previous workers (15, 27) mixed the milk preparations with either a dialyzable milk preparation or heated skimmilk]. It is noteworthy that Johnson et al. (15) observed no effect on the survival of spermatozoa of adding 50 and 100 mg per cent w/v cysteine hydrochloride to a preparation of the dialyzable component of milk. I f it is assumed that this is sufficient cysteine to detoxify skimmilk (15) by a chemical combination of its sulfhydryl groups with those in milk, it would be expected that this amount of unbound cysteine would be just as toxic to spermatozoa as unheated milk. Thus, in Johnson's study (15) perhaps cysteine did combine chemically with free sulfhydryl groups in the preparation containing the dialyzable components of milk. Thacker et al. (27) suggested that lyophilization may affect tke toxic factor(s) in milk so that this may be a reason for the absence of an over-all beneficial effect of heating the milk preparations in Experiment 3. Considering the detrimental effect on spermatozoa of a heated lactose solution (Experiment 1), and the superiority of other sugars for storing spermatozoa (18), it is suggested that it may be possible to prepare a medium from milk which is more suitable for storing spermatozoa than the standard heated milk diluents. Thus, a preparation of nondialyzable skimmilk may be expected to protect spermatozoa during dilution (17) and cooling from 30 to 5 C (4), and if lyophilized it could be stored for a considerable period. Acknowledgments

The author thanks Dr. I. C. A. Martin and Professors C. W. Emmens and R. l=f. Foote for advice and criticism. The study of ram semen was carried out at the Department of Veterinary Physiology, University of Sydney, with the aid of grants from the Australian Wool Board, and the studies of bull semen were carried out at Cornell University with grants from Eastern Artificial Insemination Coop., Inc. References

(1) Blackshaw, A. W. 1954. A bipolar rectal electrode for the electrical production of ejaculation in sheep. Australian Vet. J., 30 : 249. (2) Blackshaw, A. W. 1960. The effects of milk diluents on the viability of ram spermatozoa and their survival after freezing. Australian Vet. J., 36: 432. (3) Blackshaw, A. W., and C. W. Emmens. 1951. J . DAIRY SCIENC~ ¥O.L. 51, NO. 8

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(4)

(5)

(6)

(7) (8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

JONES The interaction of pH, osmotic pressure and electrolyte concentration on the motility of ram, bull and hmnan spermatozoa.. J. Physiol., 114: 16. Choong, C. I-I., and ]~. G. Wales. 1962. The effect of cold shock on spermatozoa. Australian J. Biol. Sci., 15: 543. Claringbold, P. J. 1957. The automatic design and analysis of biological experiments. Froc. Conf. Automatic Computation, Weapons Research Establishment, Salisbury, South Australia. Claringbold, P. J., J. D. Biggers, and C. W. Emmens. 1953. The angular transformation in quantal analysis. Biometrics, 9: 467. Collins, W. J. 1953. Evaporated milk as a semen extender. J. Dairy Sci., 36: 578. Emmens, C. W. 1947. The motility and viability of rabbit spermatozoa at different hydrogen-lon concentrations. J. Physiol., 106 : 471. Emmens, C. W., and T. J. Robinson. 1962. The semen of animals and artificial insemination. J. P. Maule, ed. Com. Agr. Bur., Bucks. p. 205. Fisher, R. A., and F. Yates. 1957. Statistical Tables for Biological, Agricultural and Medical Research. 5th ed. Oliver and Boyd, Edinburgh. Flipse, l~. J., Stuart Patton, and J. 0. Almquist. 1954. Diluents for bovine semen. I I I . Effect of lactenin and of laetoperoxidase upon spermatozoan livability. J. Dairy Sci., 37: 1205. Foote, R. H. 1962. Survival of bull sperm in milk and yolk extenders with added catalase. J. Dairy Sci., 45: 907. Jacquet, J., and R. Cassou. 1952. ~¢ouvelles reserches sur un milieu ~ base de lait 6cram6 en rue de la dilution et de la conservation du sperme de taureaux. Bull. Acad. v6t. France, 25: 149. Jaequet, J., and R. Cassou. 1952. Influence de la dilution du sperme darts tun nouveau milieu ~ base de lait ~cr6m6 sur la f6condit~ des taureaux. C. R. Acad. Agr. France, 38: 369. Johnson, P. E., R. J. Flipse, and 5. O. Almquist. 1955. Diluters for bovine semen.

J. DAIRY SCIENOE YOL. 51, NO. 8

(16)

(17)

(18)

(19)

(20) (21)

(22)

(23)

(24)

(25)

(26)

(27)

VI. The effect of eysteine hydrochlorlde on the livability of bull spermatozoa in unheated skimmilk. J. Dairy Sci., 38: 53. Jones, F. S., and It. S. Simms. 1930. The bacterial growth inhibitor (lactenin) of milk. 1. The preparation in concentrated form. J. Exptl. Med., 51: 327. Jones, R. C. 1965. Studies of the handling and insemination of ram semen. Ph.D. thesis, University of Sydney, Australia. Lapwood, K. R., and I. C. A. Martin. 1966. The use of monosaccharides, disaccharides, and trisaccharides in synthetic diluents for the storage of ram sperematozoa at 37°C and 5°C. Australian J. Biol. Sci., 19: 655. Martin, I. C. A. 1966. Diluents for the preservation of raln spermatozoa. 1. Diluents used at 37°C and 5°C containing casein. Australian J. Biol. Sci., 19: 645. Martin, I. C. A. 1965. Personal communication. Melrose, D. R. 1962. The semen of animals and artificial insemination. J. P. Maule, ed. Com. Agr. Bur., Bucks, p. 67. Patton, S. 1952. Studies of heated milk. IV. Observations on browning. J. Dairy Sci., 35: 1053. Patton, S., and R. J. Flipse. 1953. Studies of heated milk. ¥ . The reaction of lactose with milk protein as shown by lactose1-C14. J. Dairy Sci., 36: 766. (England and Wales). Report of National Institute for Research in Dairying. 1953. Shinfield, Berkshire: National Institute for Research in Dairying, University of Reading, p. 72. Saacke, 1~. G., J. O. Almquist, and R. J. Flipse. 1956. Diluters for bovine semen. VII. Effects of time and temperature of heating skimmilk upon the livability of bovine spermatozoa. J. Dairy Sci., 39: 90. Thacker, D. L., and J. O. Almquist. 1953. Diluters for bovine semen. I. Fertility and motility of bovine spermatozoa in boiled milk. J. Dairy Sci., 36: 173. Thacker, D. L., R. J. Flipse, and J. O. Almquist. 1954. Diluters for bovine semen. II. Effect of milk proteins upon spermatozoan livability. J. Dairy Sci., 37 : 220.