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Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen
Theriogenology
42:831-840,
1994
EFFECT OF TIME OF INSEMINATION RELATIVE TO OVULATION ON FERTILITY WITH LIQUID AND FROZEN BOAR SEMEN D. Waberski, K.F. Weitze, T. Gleumes, M. Schwarz, T. Willmen and R. Petzoldt Institute for Reproductive Medicine, Veterinary School of Hannc Biinteweg 15, D-30559 Hannover, Germany Received for publication: Accepted;
March 1, 1994 August 12, 1994
ABSTRACT Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=SO gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5’embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x log spermatozoa per dosage in intervals of more than 12 h before or more than 4 h, after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1 %) and liquid semen (92.5 %). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time’of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using Frozen semen. The results stress the importance of fbrther research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to es&us-weaning intervals and estrus duration. Key words: pig, insemination, ovulation
INTRODUCTION Previous experiments have shown a clear inlluence of the interval between inseminationfmating and ovulation on fertility results in swine. The highest fertility rates and numbers of accessory spermatozoa in the zona pellucida were found in sows inseminated with fresh semen close to ovulation (18). However, precise d&a on the results of per& and nostovulatory inseminations in spontaneously ovulating gilts are lacking. Therefore, the aim of Acknowledgments This study was supported by finds from the Association of German .Pig Producers ,@DS, Bonn) and the Dr. Dr. h.c. Karl Eibl Foundation. ~The authors thank Dr. Tim Baltes for reviewing the manuscript.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
832
the present study was to investigate the influence of differing time intervals relative to ovulation on fertilization rates and accessory sperm counts using liquid or frozen-thawed semen. Additionally, the extent of embryonic loss in the first 28 d of pregnancy following pre- and postovulatory inseminations with liquid semen was studied. MATERIALS AND METHODS Animals, Estrus, and Ovulation Detection Gilts (Experiment 1: n=76, Experiment 2: n=SO, Experiment 3: n=24) were housed in groups of 2 to 5 with visual and olfactory boar contact. Estrus detection was performed in 12-h (Experiments 1 and 3) or 8-h intervals (Experiment 2) using a teaser boar. Sows were considered to be in estrus when they stood to be mounted. During estrus, transcutaneous sonography of ovaries was performed every 4 h using a 5MHz sector scanner (Sonoline SX, Siemens Company, Germany) for determination of the time of ovulation, as described previously (19). Semen In Experiments 1 and 3, mixed semen from 3 boars diluted to 2 x 109 spermatozoa per dosage with Androhep medium (Minitube Company; 17) was used for insemination within 48 h after collection. In Experiment 2, the semen of 4 boars was frozen on a split sample basis as described previously (23) according the modified Westendorf procedure in macrotubes or flat straws. The thawed semen was diluted with 80 ml Glucose-EDTA-extender (MerckR Minitube Company, Landshut, Germany) and used for insemination at a dosage of 5 x 109 spermatozoa. Insemination In Experiment 1, 25 gilts were inseminated once 24 h after detecting boar tolerance (preovulatory group), and 51 gilts were inseminated either 0, 4, or 8 h after the detection of ovulation (Figure 1). Using frozen-thawed semen (Experiment 2) gilts were inseminated once either at 36 h (n=22), 40 h (n=21), or 44 h (n=21) after the detection of boar tolerance, and 16 gilts were inseminated diiectly afler detection of ovulation. In Experiment 3, gilts were inseminated once at 32 h (n=9) after the detection of boar tolerance or at 0 h (n=lO), or 4 h (n=5) after the detection of ovulation. In all experiments the interval between AI and ovulation in preovulatoty inseminated gilts was calculated retrospectively after the detection of ovulation. Fertilization Status In Experiments 1 and 2, embryos and oocytes were flushed from the genital tracts of gilts slaughtered 2 to 5 d after insemination. Quality assessment of the embryos was based on the following criteria: 1) the presence of an even perivitelline space with evenly distributed blastomeres, 2) the presence of accessory spermatozoa in the zona pellucida, and 3) developmental stages corresponding to the time between ovulation and flushing. Multicellular structures not fulfilhng Criteria 1 and/or 3 were classified as degenerated. The recovery rate was determined as ihe percentage of embryos plus oocytes recovered based on the corpora lutea count. The pregnancy rate was calculated aa the percentage of gilts with at least 1 normally developed embryo. The fertiliition rate was calculated as the number of normally developed embryos divided by the total number of embryos and oocytes. The nuclei and the accessory
833
Theriogenology
spermatozoa in the zonae pellucidae were counted after staining the embryosfoocytes in a solution containing 25 ug of the DNA stain Hoechst 33342 (Sigma Chemicals Company, Deisenhofen, Germany) per ml PBS. A fluorescence microscope with the excitation filter G365, the dichroic beam splitter FT395, and the emission filter LP420 was used at x 400 magnification. A nuclei count of 0 or 1 was wnsidered an unfertilized oocyte. In Experiment 3, gilts were slaughtered 28 d after insemination and the genital tracts were removed. The number of corpora lutea on each ovary was counted, as was the number of conceptuses in each uterine horn. The length and form of the embryos were determined. Embryos with a crown-rump length of at least 1.8 cm and with the rudimentary eyes, extremities, and hearts were considered to be normally developed.
Folliclesa +
+
+
+
+
I Time of AI
I
I
I
? 16-12
f 12-8
t 8-4
? 4-o
? o-4
,r 4-8
I ? 8-12
7
7
6
18
17
16
Ovulation
I
Interval (hours)Aldov Gilts (n)
5
a Follicles visible (+) or not visible (-) using transcutaneous sonography in 4-hour intervals. b In preovulatory inseminated gilts, the interval between insemination (AI) and ovulation (OV) was calculated retrospectively (insemination 24 hours after the detection of boar tolerance). Figure 1. Determination
of time intervals of insemination relative to ovulation in Experiment 1
Statistical Analysis Calculations were made using the SAS program (14). The influence of the fixed effects classified time.intervals of insemination relative to ovulation (Experiments 1 and 3), respective classified time intervals of insemination relative to ovulation, semen packaging, and boar (Experiment 2) were estimated by analysis of variance. The frequencies of pregnant gilts and unilateral fertilization distributed to the time intervals of insemination relative to ovulation were tested using C&square contingency tables. The means for the fertilization rates were compared using the t-test. In Experiments 1 and 2, accessory sperm data (number of spermatozoa per zona pellucida of embryos and oocytes) was tirst transformed by the equation log10 (x+1). This step was performed to reduce the large and highly skewed numbers of accessory spermatozoa per zona. The means for the classified insemination time intervals and the means for the percentage of zonae with accessory spermatozoa were compared using the t-test. Zonae pellucidae were divided into 4 classes according the numbers of accessory spermatozoa: 1) 0 to 10, 2) 11 to 40, 3) 41 to 80, 4) > 80 (Experiment l), and 1) 0 to 10, 2) 11 to 20, 3) 21 to 40, 4) > 40 (Experiment 2), respectively. The frequency classes of zonae distributed to classified time intervals of insemination were compared using Chi-square contingency tables.
834
Theriogenology
RESULTS Experiment 1 The recovery rate of embryos and oocytes relative to the number of corpora lutea averaged 92.1 % and did not vary significantly between the insemination groups. Fertilization data is shown in Table 1. The pregnancy rate was 100 % in gilts inseminated in the interval 12 h before to 4 h after ovulation; the ditTerences to the other insemination groups were not significant. The fertilization rate decreased significantly (PcO.05) in the gilts inseminated more than 12 h before or more than 4 h after ovulation. The incidence of unilateral fertilization was 20 % in the preovulatory AI group with an insemination-ovulation interval of 16 to 12 h, and 31.3 % in the group inseminated 8 to 12 h after detection of ovulation. Accessory sperm data is shown in Table 2. Average numbers of accessory spermatozoa per embryo or oocyte were significantly higher (PcO.05) in the AI groups 8 to 4 h and 4 to 0 h before, and 0 to 4 h after ovulation compared to the other groups. Frequency distributions of accessory sperm data showed a significant shit? between all insemination groups (FcO.05). Gilts inseminated 4 to 0 h before the detection of ovulation had significantly highest numbers of embryos in the high accessory sperm number classes (41 to 80 and > 80 accessory spermatozoa per embryo) compared to the other groups of gilts. Experiment 2 The recovery rate of embryos and oocytes averaged 86.7 % and did not vary significantly between the insemination groups. No influence of individual boars and packaging on fertility results was seen. Fertilization data is shown in Table 3. Both pregnancy and fertilization rate were significantly higher (PcO.05) in gilts inseminated in the interval 4 to 0 h before ovulation (100 and 88.1 %, respectively), than in the other groups. No fertilization occured when insemination was performed more than 8 h before ovulation. Accessory sperm data (Table 4) showed the highest average number of spermatozoa per zona, the highest percentage of zonae having at least one accessory spermatozoon, and a significant shift to classes of higher numbers (21 to 40, >40) of accessory spermatzoa per embryo in the group inseminated 0 to 4 h before ovulation (PcO.05). No accessory spermatozoa were detected following AI more than 8 h before ovulation. Experiment 3 The number of embryos per gilt and the fertilization rate of Day 28-embryos differed significantly between preovulatory and postovulatory inseminations (PcO.05). The pregnancy rate did not differ significantly between the groups (Table 5). DISCUSSION Successful fertilization depends mainly on the time of insemination/mating relative to ovulation. Previous studies with hCG-treated sows have shown that insemination close to ovulation leads to the highest fertility results, which is especially important when frozen semen is used (10). In the present experiments spontaneously cycling and ovulating gilts together with a precise and stress tree technique of detection of ovulation were used. Since reliable data on fertilization in preovulatory-inseminated gilts exists (18), special emphasis was laid on peri- and
835
Theriogenology
Table 1. Pregnancy rate, fertikation rate, and unilateral fertilization following pre- and postovulatory insemination using liquid boar semen (Experiment 1)
Interval AI-ov hours
Gilts n
Embryos/ Embryos oocytes normal degenerated n n n
Preovulatory insemination 5 58 16-12 7 76 12-8 7 82 8-4 6 71 4-O Postovulatory insemination 18 214 O-4 17 209 4-8 16 190 8-12 a-cValues with diierent OV = ovulation.
Oocytes Pregnancy rate n %
Fertilization rate %
Unilateral fertilization %
20.0a
32 73 70 67
0 3 5 2
26 0 7 2
80.0a 1ooa
55.8a 96.ob
looa looa
85sb 93.4b
198 149 112
3 21 13
13 39 65
looa
92.+
ma
71.4c 58.9a
5.8a 31.3a
82.5a 75.0a
oa i4.3a
16.6a
superscripts within columns differ significantly (PcO.05).
Table 2. Number of accessory spermatozoa in the zonae of embryos and oocytes following pre- and postovulatory insemination using liquid boar semen (Experiment 1)
Interval AI-ov hours
Gilts Zonae Accessory spermatozoa Zonae with Frequency (%) of accessory per zona accessory spermatozoa sperm number classes n n j7 SD % O-10 11-40 41-80 >XO
Preovulatory insemination 5 58 10.9a 16-12 7 76 58.6a,b 12-8 7 82 124.7bc 8-4 6 71 201.7c 4-O Postovulatory insemination 214 127.lc.b O-4 18 209 75.5qb 4-8 17 190 75.3ab 8-12 16
10.5 46.9 114.7 126.9
56.9a loob 86.6b 97.2b
63.8 27.6 26.8 2.8
32.8 23.8 9.8 9.8
3.4 22.4 15.9 16.9
104.9 105.1 71.7
97.2b 76.lc 60.5a
8.4 35.9 42.9
21.1 27.7 8.4
17.8 11.0 6.3
a-cValues with different supercripts within columns differ significantly (PxO.05). dj DiBerent scripts indicate significant differences in the distribution of the accessory sperm number classes (PcO.05). OV = ovulation.
Od 26.3e 47.5f 70.58 52.8h 25.4i 42.61’
836
Theriogenology
Table 3. Pregnancy rate, fertilization rate, and unilateral fertilization following pre- and postovulatory insemination using frozen boar semen (Experiment 2)
Interval AI-ov hours
Gilts n
Embryos/ Embryos oocytes normal degenerated n n n
Preovulatory insemination 16 182 >8 24 264 8-4 24 261 4-O Postovulatory insemination 16 190 O-4
Oocytes Pregnancy rate n %
0 145 230
0 15 11
182 104 20
95
30
65
Fertilization rate %
Unilateral fertilization %
oa 7o.sb
oa 54.9b
16.7a
1ooc
88.lC
8.3a
68.8b
so.ob
18.7a
a-Walues with different superscripts within columns differ significantly (RO.05). OV = ovulation.
Table 4. Number of accessory spermatozoa in the zonae of embryos and oocytes following pre- and postovulatory insemination using Cozen boar semen (Experiment 2)
Interval AI-ov hours
Gilts Zonae Accessory spermatozoa per zona F n n SD
Preovulatory insemination 182 Oa >8 16 264 13.lb 8-4 24 . *. Po4s;oowlat?q mszLtron285c O-4
16
190
4.5d
Zonae with Frequency (“h) of accessory sperm number classes accessory spermatozoa % O-10 II-20 21-40 > 40
oa
100
0 27.4 32.9
65.2b 96.9c
70.8 30.7
10.6
6o.ob
92.1
0
0
oe
14.0 6.4 8.7f 32.2 15.3 21.88 2.1
2.1
a-dValues with diflkrent supercripts within columns diier significantly (PCO.05). e-g Different scripts indicate significant differences in the distribution of the accessory sperm number classes (PcO.05). OV = ovulation.
3.7f
837
Theriogenology
Table 5. Number of Day-28 embryos per gilt, pregnancy rate, and fertilization rate following pre- and postovulatory insemination using liquid boar semen (Experiment 3)
Interval AI-ov hours
Gilts n
C. lutea per gilt 0
Preovulatory insemination 16-O 9 13.4a Postovulatory insemination o-4 10 12.9a 4-8 5 14.2a a,bValues with diierent OV = ovulation.
Embryos per gilt 0
Length of embryos Pregnancy rate
Fertilization rate
cm
%
%
10.1a
2.2a
88.9a
75.2a
7.7b 7.8b
2.3a 2.3a
80.0a 1ooa
59.71) 54.9b
superscripts within columns differ significantly (PcO.05).
postovulatory insemination results in this study . Regarding the initial pregnancy and fertilization rates, the optimal timing of AI using liquid semen was within a relatively short period of 12 h before to 4 h after ovulation (Experiment 1). Using frozen semen (Experiment 2) initial pregnancy and fertilization rates similar to those following AI with liquid semen could be obtained even when a single insemination was performed, if the interval between insemination and ovulation was at maximum 4 h (Figure 2). The fertilization rate (88.1 %) in this AI group exceeds fertilization rates which can be expected using frozen boar semen under controlled field conditions (1,9) by 20 to 30 %. The strong decrease of fertilization rates with increasing intervals between insemination and ovulation after use of frozen semen confirm the reduced viability of frozen-thawed spermatozoa in the female genital tract compared to liquid semen as described in earlier studies (10,11,13). In Experiment 1 gilts inseminated 8 to 12 h after ovulation showed an enhanced, statistically insignificant increase in the incidence of unilateral fertilization to 31.3 %; under regular AI conditions the incidence is below 10 % (5). This is explained by an increase of unilateral sperm transport to one oviduct due to decreasing estradiol and increasing progesterone levels at late estrus (5,7). The viable We span of pig oocytes in the fallopian tube is some 8 to 10 h, but oocytes fertilized at even this short interval after ovulation appear less likely to form a viable embryo (Hunter 5,6). The reduced viability of pig embryos after postovulatory insemination is attributed to an increased incidence of polyspermic fertilization. The nature of the postovulatory degeneration that enhances an oocyte’s susceptibility to polyspermic fertilization has not yet been clarified, but it is suspected to involve vitelline organelles and the plasmalemma. However, the principle factor is considered to originate from increased numbers of spermatozoa reaching the site of fertilization due to the above mentioned changes in hormonal activity at late estrus (8). In the present experiments 1 and 2 the incidence of polyspermic fertilization could not be evaluated, since polyspermic-fertilized oocytes are able to undergo apparently normal development at least until the 4-to S-cell stage (5). However, the evaluation of Day-28 embryos in Experiment 3 revealed that postovulatory insemination, even in the short interval 0 to 4 h after ovulation, led to
838
Theriogenology Fertilization
rate
(%I
-._
0vul;tion
n
80
80
18-12
4-o relative
8-4 12-8 Interval of insemination m
Liquid
semen
o-4 . to ovulation
b$$?$!Frozen
4,?
_ 8-12
tnours)
Semen
Figure 2. Fertilization rates (%) following diiering times of insemination relative to ovulation using liquid (Experiment 1; n=76) or frozen (Experiment 2; n=SO) semen.
ACCe88Ory 250
spermatozoa
(3 Ovulation 201.7
w-12
12-8 8-4 Interval of inseminatio~i -
Liquid
semen
4-o relative m
o-4 to ovulation Frozen
4-8 (hours?
8-12
semen
Figure 3. Mean number of accessory spermatozoa in zonae of embryos and oocytes following differing times of insemination relative to ovulation using liquid (Experiment 1; n=76) or frozen (Experiment 2; n=SO) semen.
839
Theriogenology
increased rates of embryonic loss during early pregnancy, compared to preovulatory inseminated gilts. Recently it was demonstrated that delayed mating in relation to ovulation decreased mean blastocyst size and estrogen secretion in vitro when blastocysts were collected 288 h post-hCG (2). The decrease in fertilization rates following postovulatory insemination resulted from an increased number of unfertilized ova while the percentage of degenerated embryos remained constant (Experiment 1). Together with the findings of decreased numbers of accessory spermatozoa it can be suggested that the decrease in fertilization rate in delayed inseminated gilts is due to deficiencies in sperm transport as reported earlier by Hunter (5). The more striking decline of pregnancy and fertilization rates after postovulatory insemination with frozen semen compared to liquid semen can be explained by a higher elimination rate in the female genital tract due to cell damage after cryopreservation, resulting in lower sperm numbers at the site of fertilization (11,lS). This was even obvious in the optimal insemination interval close to ovulation, where initial pregnancy and fertilization rates were similar between frozen and liquid semen: the numbers of accessory spermatozoa were ten-times lower using frozen semen compared to liquid semen (Figure 3), although a higher dosage of frozen spermatozoa (5 x log vs 2 x log) was used. In contrast to previous experiments (16,21), no differences in fertilization results were found between frozen semen in round and flat straws. The reason for this is not clear. It might be that in the present study the different intervals of insemination relative to ovulation were the dominating factor influencing the fertilization capacity of frozen semen. In conclusion, the optimal time of insemination using liquid semen is in an interval from 12 to 0 h before ovulation. With frozen semen fertilization results not diiering from those using liquid semen can be achieved when insemination is performed in an interval of approximately 4 h before ovulation. The low tolerance for the optimal timing of AI indicates the importance of tkther studies on sperm transport and ovulation stimulating substances present in seminal plasma (3,4,12,20). In addition, the recently described relationship between the onset of estrus after weaning, the duration of estrus and the time of ovulation (22) offers chances for successful fertiliition using frozen semen in optimally controlled sow herds. REFERENCES 1. Bwanga CO, Hofino PO, Grevle IS, Einarsson S, Rodriguez-Martinez H. In vivo fertilizing capacity of deep frozen boar semen packaged in plastic bags and maxi-straws. J Vet Med A 1991;38:281-286. 2. Ckrdenas II, Pope WF. Effect of time of mating relative to ovulation on morpholgical diversity of swine blastocysts. Biol Reprod 1993;49:1015-1018. 3. Claus R. Physiological role of seminal plasma components in the female reproductive tract. J Reprod Fertil 1990; 40 (Suppl): 117- 13 1. 4. Einarsson S, Viring S. Distribution of frozen-thawed spermatozoa in the reproductive tract of gilts at different time intervals after insemination. J Reprod Fertil 1973;32:117-120. 5. Hunter RHF. The effects of delayed insemination on fertilization and early cleavage in the pig. J Reprod Fertil 1967;13: 133-147. 6. Hunter RHF. Physiological factors influencing ovulation, fertilization, early embryonic development and establishment of pregnancy in pigs. Br Vet J 1977;133:461-470.
840 7. Hunter RHF. Interrelationships
8. 9.
10.
11.
12.
13. 14. 15.
16. 17. 18.
19. 20.
21.
22.
23.
Theriogenology
between spermatozoa, the female reproductive tract, and the egg investments. In: Cole DJA, Foxcroft GR (eds), Control of Pig Reproduction, Buttersworth Scientilic, London, 1982; 49-63. Hunter RI-IF. Oviduct iimction in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991;29:385-39i. Johnson LA Fertility results using frozen boar semen: 1970 to 1985. In: Johnson LA, Larsson K (eds), Deep Freezing of Boar Semen, Uppsala, Sweden, 1985; 199-222. Larsson K. Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation. Intluence of boars and thawing diluents. Acta Vet Stand 1976;17:63-73. Purse1 VG, Schulmann LL, Johnson LA. Distribution and morphology of fresh and frozen thawed sperm in the reproductive tract of gilts after artificial insemination. Biol Reprod 1978;19:69-76. Rath D, Weitze KF, Pena Alfaro CE, Andrade Moura JC. Effects of seminal plasma on the number of accessory sperm cells and fertilization in gilts. Zuchthygiene 1989;24: 123127. Saacke RG. Semen quality in relation to semen preservation. J Dairy Sci 1982;66:26352644. SAS User’s Guide: Statistics SAS Institute, Inc, Cary, 1985. Scheffels W, Biegert W, Leidl W. Die Verteihmg der Spermien im weiblichen Genitale des Schweines nach Insemination von fXschem und tiefgefrorenen Sperma. Zuchthygiene 1971;6:60-64. Simmet C, Gall T, Waberski D, Weitze KF. Fertility of boar semen frozen in flat straws and macrotubes. Reprod Dom Anim 1993;2 (Suppl):87 abstr. Waberski D, Weitze KF, Rath D, Salhnann HP. Wnkung von bovinem Serumalbumin und Zwitterionenpuffer auf fliissigkonservierten Ebersamen. Zuchthygiene 1989;24: 128-133. Waberski D, Weitze KF, Lietmann C, Ltibbert zur Lage W, Bortolozzo FP, Wilhnen T, Petzoldt R The initial fertilizing capacity of longterm-stored liquid boar semen following pre- and postovulatory insemination. Theriogenology 1994;4 1: 1367- 1377. Weitze KF, Habeck 0, Willmen T, Rath D. Detection of ovulation in the sow using transcutaneous sonography. Zuchthygiene 1989; 24140-42. Weitze KF, Lotz JH, Everwand A, Willmen T, Waberski D. Interaction between inseminate, uterine and ovarial fimction in the sow. II. Investigations into the influencing of ovulation by the use of sperm-&e media. Reprod Dom Anim 1990;25: 197-204. Weitze KF, Stampa E, Richter L, Wdhnen T, Waberski D. Fertility of frozen boar semen: influence of packaging, number of inseminations, and seminal plasma. Reprod Dom Anim 1991;l (Suppl):139-142. Weitze KF, Waberski D, Wagner-Rietschel II, Richter L, Kalm E. The onset of heat after weaning, heat duration, and ovulation as major factors in AI timing in sows. Reprod Dom Anim (m press). WestendorfP, Richter L, Treu H. Zur Tiefgefiierung von Ebersperma: Labor- und Besamungsergebnisse mit dem Htilsenberger Pailletten-Verfahren. Dtsch tier&rztl Wochenschr 1975;82,261-267.
42:831-840,
1994
EFFECT OF TIME OF INSEMINATION RELATIVE TO OVULATION ON FERTILITY WITH LIQUID AND FROZEN BOAR SEMEN D. Waberski, K.F. Weitze, T. Gleumes, M. Schwarz, T. Willmen and R. Petzoldt Institute for Reproductive Medicine, Veterinary School of Hannc Biinteweg 15, D-30559 Hannover, Germany Received for publication: Accepted;
March 1, 1994 August 12, 1994
ABSTRACT Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=SO gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5’embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x log spermatozoa per dosage in intervals of more than 12 h before or more than 4 h, after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1 %) and liquid semen (92.5 %). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time’of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using Frozen semen. The results stress the importance of fbrther research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to es&us-weaning intervals and estrus duration. Key words: pig, insemination, ovulation
INTRODUCTION Previous experiments have shown a clear inlluence of the interval between inseminationfmating and ovulation on fertility results in swine. The highest fertility rates and numbers of accessory spermatozoa in the zona pellucida were found in sows inseminated with fresh semen close to ovulation (18). However, precise d&a on the results of per& and nostovulatory inseminations in spontaneously ovulating gilts are lacking. Therefore, the aim of Acknowledgments This study was supported by finds from the Association of German .Pig Producers ,@DS, Bonn) and the Dr. Dr. h.c. Karl Eibl Foundation. ~The authors thank Dr. Tim Baltes for reviewing the manuscript.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
832
the present study was to investigate the influence of differing time intervals relative to ovulation on fertilization rates and accessory sperm counts using liquid or frozen-thawed semen. Additionally, the extent of embryonic loss in the first 28 d of pregnancy following pre- and postovulatory inseminations with liquid semen was studied. MATERIALS AND METHODS Animals, Estrus, and Ovulation Detection Gilts (Experiment 1: n=76, Experiment 2: n=SO, Experiment 3: n=24) were housed in groups of 2 to 5 with visual and olfactory boar contact. Estrus detection was performed in 12-h (Experiments 1 and 3) or 8-h intervals (Experiment 2) using a teaser boar. Sows were considered to be in estrus when they stood to be mounted. During estrus, transcutaneous sonography of ovaries was performed every 4 h using a 5MHz sector scanner (Sonoline SX, Siemens Company, Germany) for determination of the time of ovulation, as described previously (19). Semen In Experiments 1 and 3, mixed semen from 3 boars diluted to 2 x 109 spermatozoa per dosage with Androhep medium (Minitube Company; 17) was used for insemination within 48 h after collection. In Experiment 2, the semen of 4 boars was frozen on a split sample basis as described previously (23) according the modified Westendorf procedure in macrotubes or flat straws. The thawed semen was diluted with 80 ml Glucose-EDTA-extender (MerckR Minitube Company, Landshut, Germany) and used for insemination at a dosage of 5 x 109 spermatozoa. Insemination In Experiment 1, 25 gilts were inseminated once 24 h after detecting boar tolerance (preovulatory group), and 51 gilts were inseminated either 0, 4, or 8 h after the detection of ovulation (Figure 1). Using frozen-thawed semen (Experiment 2) gilts were inseminated once either at 36 h (n=22), 40 h (n=21), or 44 h (n=21) after the detection of boar tolerance, and 16 gilts were inseminated diiectly afler detection of ovulation. In Experiment 3, gilts were inseminated once at 32 h (n=9) after the detection of boar tolerance or at 0 h (n=lO), or 4 h (n=5) after the detection of ovulation. In all experiments the interval between AI and ovulation in preovulatoty inseminated gilts was calculated retrospectively after the detection of ovulation. Fertilization Status In Experiments 1 and 2, embryos and oocytes were flushed from the genital tracts of gilts slaughtered 2 to 5 d after insemination. Quality assessment of the embryos was based on the following criteria: 1) the presence of an even perivitelline space with evenly distributed blastomeres, 2) the presence of accessory spermatozoa in the zona pellucida, and 3) developmental stages corresponding to the time between ovulation and flushing. Multicellular structures not fulfilhng Criteria 1 and/or 3 were classified as degenerated. The recovery rate was determined as ihe percentage of embryos plus oocytes recovered based on the corpora lutea count. The pregnancy rate was calculated aa the percentage of gilts with at least 1 normally developed embryo. The fertiliition rate was calculated as the number of normally developed embryos divided by the total number of embryos and oocytes. The nuclei and the accessory
833
Theriogenology
spermatozoa in the zonae pellucidae were counted after staining the embryosfoocytes in a solution containing 25 ug of the DNA stain Hoechst 33342 (Sigma Chemicals Company, Deisenhofen, Germany) per ml PBS. A fluorescence microscope with the excitation filter G365, the dichroic beam splitter FT395, and the emission filter LP420 was used at x 400 magnification. A nuclei count of 0 or 1 was wnsidered an unfertilized oocyte. In Experiment 3, gilts were slaughtered 28 d after insemination and the genital tracts were removed. The number of corpora lutea on each ovary was counted, as was the number of conceptuses in each uterine horn. The length and form of the embryos were determined. Embryos with a crown-rump length of at least 1.8 cm and with the rudimentary eyes, extremities, and hearts were considered to be normally developed.
Folliclesa +
+
+
+
+
I Time of AI
I
I
I
? 16-12
f 12-8
t 8-4
? 4-o
? o-4
,r 4-8
I ? 8-12
7
7
6
18
17
16
Ovulation
I
Interval (hours)Aldov Gilts (n)
5
a Follicles visible (+) or not visible (-) using transcutaneous sonography in 4-hour intervals. b In preovulatory inseminated gilts, the interval between insemination (AI) and ovulation (OV) was calculated retrospectively (insemination 24 hours after the detection of boar tolerance). Figure 1. Determination
of time intervals of insemination relative to ovulation in Experiment 1
Statistical Analysis Calculations were made using the SAS program (14). The influence of the fixed effects classified time.intervals of insemination relative to ovulation (Experiments 1 and 3), respective classified time intervals of insemination relative to ovulation, semen packaging, and boar (Experiment 2) were estimated by analysis of variance. The frequencies of pregnant gilts and unilateral fertilization distributed to the time intervals of insemination relative to ovulation were tested using C&square contingency tables. The means for the fertilization rates were compared using the t-test. In Experiments 1 and 2, accessory sperm data (number of spermatozoa per zona pellucida of embryos and oocytes) was tirst transformed by the equation log10 (x+1). This step was performed to reduce the large and highly skewed numbers of accessory spermatozoa per zona. The means for the classified insemination time intervals and the means for the percentage of zonae with accessory spermatozoa were compared using the t-test. Zonae pellucidae were divided into 4 classes according the numbers of accessory spermatozoa: 1) 0 to 10, 2) 11 to 40, 3) 41 to 80, 4) > 80 (Experiment l), and 1) 0 to 10, 2) 11 to 20, 3) 21 to 40, 4) > 40 (Experiment 2), respectively. The frequency classes of zonae distributed to classified time intervals of insemination were compared using Chi-square contingency tables.
834
Theriogenology
RESULTS Experiment 1 The recovery rate of embryos and oocytes relative to the number of corpora lutea averaged 92.1 % and did not vary significantly between the insemination groups. Fertilization data is shown in Table 1. The pregnancy rate was 100 % in gilts inseminated in the interval 12 h before to 4 h after ovulation; the ditTerences to the other insemination groups were not significant. The fertilization rate decreased significantly (PcO.05) in the gilts inseminated more than 12 h before or more than 4 h after ovulation. The incidence of unilateral fertilization was 20 % in the preovulatory AI group with an insemination-ovulation interval of 16 to 12 h, and 31.3 % in the group inseminated 8 to 12 h after detection of ovulation. Accessory sperm data is shown in Table 2. Average numbers of accessory spermatozoa per embryo or oocyte were significantly higher (PcO.05) in the AI groups 8 to 4 h and 4 to 0 h before, and 0 to 4 h after ovulation compared to the other groups. Frequency distributions of accessory sperm data showed a significant shit? between all insemination groups (FcO.05). Gilts inseminated 4 to 0 h before the detection of ovulation had significantly highest numbers of embryos in the high accessory sperm number classes (41 to 80 and > 80 accessory spermatozoa per embryo) compared to the other groups of gilts. Experiment 2 The recovery rate of embryos and oocytes averaged 86.7 % and did not vary significantly between the insemination groups. No influence of individual boars and packaging on fertility results was seen. Fertilization data is shown in Table 3. Both pregnancy and fertilization rate were significantly higher (PcO.05) in gilts inseminated in the interval 4 to 0 h before ovulation (100 and 88.1 %, respectively), than in the other groups. No fertilization occured when insemination was performed more than 8 h before ovulation. Accessory sperm data (Table 4) showed the highest average number of spermatozoa per zona, the highest percentage of zonae having at least one accessory spermatozoon, and a significant shift to classes of higher numbers (21 to 40, >40) of accessory spermatzoa per embryo in the group inseminated 0 to 4 h before ovulation (PcO.05). No accessory spermatozoa were detected following AI more than 8 h before ovulation. Experiment 3 The number of embryos per gilt and the fertilization rate of Day 28-embryos differed significantly between preovulatory and postovulatory inseminations (PcO.05). The pregnancy rate did not differ significantly between the groups (Table 5). DISCUSSION Successful fertilization depends mainly on the time of insemination/mating relative to ovulation. Previous studies with hCG-treated sows have shown that insemination close to ovulation leads to the highest fertility results, which is especially important when frozen semen is used (10). In the present experiments spontaneously cycling and ovulating gilts together with a precise and stress tree technique of detection of ovulation were used. Since reliable data on fertilization in preovulatory-inseminated gilts exists (18), special emphasis was laid on peri- and
835
Theriogenology
Table 1. Pregnancy rate, fertikation rate, and unilateral fertilization following pre- and postovulatory insemination using liquid boar semen (Experiment 1)
Interval AI-ov hours
Gilts n
Embryos/ Embryos oocytes normal degenerated n n n
Preovulatory insemination 5 58 16-12 7 76 12-8 7 82 8-4 6 71 4-O Postovulatory insemination 18 214 O-4 17 209 4-8 16 190 8-12 a-cValues with diierent OV = ovulation.
Oocytes Pregnancy rate n %
Fertilization rate %
Unilateral fertilization %
20.0a
32 73 70 67
0 3 5 2
26 0 7 2
80.0a 1ooa
55.8a 96.ob
looa looa
85sb 93.4b
198 149 112
3 21 13
13 39 65
looa
92.+
ma
71.4c 58.9a
5.8a 31.3a
82.5a 75.0a
oa i4.3a
16.6a
superscripts within columns differ significantly (PcO.05).
Table 2. Number of accessory spermatozoa in the zonae of embryos and oocytes following pre- and postovulatory insemination using liquid boar semen (Experiment 1)
Interval AI-ov hours
Gilts Zonae Accessory spermatozoa Zonae with Frequency (%) of accessory per zona accessory spermatozoa sperm number classes n n j7 SD % O-10 11-40 41-80 >XO
Preovulatory insemination 5 58 10.9a 16-12 7 76 58.6a,b 12-8 7 82 124.7bc 8-4 6 71 201.7c 4-O Postovulatory insemination 214 127.lc.b O-4 18 209 75.5qb 4-8 17 190 75.3ab 8-12 16
10.5 46.9 114.7 126.9
56.9a loob 86.6b 97.2b
63.8 27.6 26.8 2.8
32.8 23.8 9.8 9.8
3.4 22.4 15.9 16.9
104.9 105.1 71.7
97.2b 76.lc 60.5a
8.4 35.9 42.9
21.1 27.7 8.4
17.8 11.0 6.3
a-cValues with different supercripts within columns differ significantly (PxO.05). dj DiBerent scripts indicate significant differences in the distribution of the accessory sperm number classes (PcO.05). OV = ovulation.
Od 26.3e 47.5f 70.58 52.8h 25.4i 42.61’
836
Theriogenology
Table 3. Pregnancy rate, fertilization rate, and unilateral fertilization following pre- and postovulatory insemination using frozen boar semen (Experiment 2)
Interval AI-ov hours
Gilts n
Embryos/ Embryos oocytes normal degenerated n n n
Preovulatory insemination 16 182 >8 24 264 8-4 24 261 4-O Postovulatory insemination 16 190 O-4
Oocytes Pregnancy rate n %
0 145 230
0 15 11
182 104 20
95
30
65
Fertilization rate %
Unilateral fertilization %
oa 7o.sb
oa 54.9b
16.7a
1ooc
88.lC
8.3a
68.8b
so.ob
18.7a
a-Walues with different superscripts within columns differ significantly (RO.05). OV = ovulation.
Table 4. Number of accessory spermatozoa in the zonae of embryos and oocytes following pre- and postovulatory insemination using Cozen boar semen (Experiment 2)
Interval AI-ov hours
Gilts Zonae Accessory spermatozoa per zona F n n SD
Preovulatory insemination 182 Oa >8 16 264 13.lb 8-4 24 . *. Po4s;oowlat?q mszLtron285c O-4
16
190
4.5d
Zonae with Frequency (“h) of accessory sperm number classes accessory spermatozoa % O-10 II-20 21-40 > 40
oa
100
0 27.4 32.9
65.2b 96.9c
70.8 30.7
10.6
6o.ob
92.1
0
0
oe
14.0 6.4 8.7f 32.2 15.3 21.88 2.1
2.1
a-dValues with diflkrent supercripts within columns diier significantly (PCO.05). e-g Different scripts indicate significant differences in the distribution of the accessory sperm number classes (PcO.05). OV = ovulation.
3.7f
837
Theriogenology
Table 5. Number of Day-28 embryos per gilt, pregnancy rate, and fertilization rate following pre- and postovulatory insemination using liquid boar semen (Experiment 3)
Interval AI-ov hours
Gilts n
C. lutea per gilt 0
Preovulatory insemination 16-O 9 13.4a Postovulatory insemination o-4 10 12.9a 4-8 5 14.2a a,bValues with diierent OV = ovulation.
Embryos per gilt 0
Length of embryos Pregnancy rate
Fertilization rate
cm
%
%
10.1a
2.2a
88.9a
75.2a
7.7b 7.8b
2.3a 2.3a
80.0a 1ooa
59.71) 54.9b
superscripts within columns differ significantly (PcO.05).
postovulatory insemination results in this study . Regarding the initial pregnancy and fertilization rates, the optimal timing of AI using liquid semen was within a relatively short period of 12 h before to 4 h after ovulation (Experiment 1). Using frozen semen (Experiment 2) initial pregnancy and fertilization rates similar to those following AI with liquid semen could be obtained even when a single insemination was performed, if the interval between insemination and ovulation was at maximum 4 h (Figure 2). The fertilization rate (88.1 %) in this AI group exceeds fertilization rates which can be expected using frozen boar semen under controlled field conditions (1,9) by 20 to 30 %. The strong decrease of fertilization rates with increasing intervals between insemination and ovulation after use of frozen semen confirm the reduced viability of frozen-thawed spermatozoa in the female genital tract compared to liquid semen as described in earlier studies (10,11,13). In Experiment 1 gilts inseminated 8 to 12 h after ovulation showed an enhanced, statistically insignificant increase in the incidence of unilateral fertilization to 31.3 %; under regular AI conditions the incidence is below 10 % (5). This is explained by an increase of unilateral sperm transport to one oviduct due to decreasing estradiol and increasing progesterone levels at late estrus (5,7). The viable We span of pig oocytes in the fallopian tube is some 8 to 10 h, but oocytes fertilized at even this short interval after ovulation appear less likely to form a viable embryo (Hunter 5,6). The reduced viability of pig embryos after postovulatory insemination is attributed to an increased incidence of polyspermic fertilization. The nature of the postovulatory degeneration that enhances an oocyte’s susceptibility to polyspermic fertilization has not yet been clarified, but it is suspected to involve vitelline organelles and the plasmalemma. However, the principle factor is considered to originate from increased numbers of spermatozoa reaching the site of fertilization due to the above mentioned changes in hormonal activity at late estrus (8). In the present experiments 1 and 2 the incidence of polyspermic fertilization could not be evaluated, since polyspermic-fertilized oocytes are able to undergo apparently normal development at least until the 4-to S-cell stage (5). However, the evaluation of Day-28 embryos in Experiment 3 revealed that postovulatory insemination, even in the short interval 0 to 4 h after ovulation, led to
838
Theriogenology Fertilization
rate
(%I
-._
0vul;tion
n
80
80
18-12
4-o relative
8-4 12-8 Interval of insemination m
Liquid
semen
o-4 . to ovulation
b$$?$!Frozen
4,?
_ 8-12
tnours)
Semen
Figure 2. Fertilization rates (%) following diiering times of insemination relative to ovulation using liquid (Experiment 1; n=76) or frozen (Experiment 2; n=SO) semen.
ACCe88Ory 250
spermatozoa
(3 Ovulation 201.7
w-12
12-8 8-4 Interval of inseminatio~i -
Liquid
semen
4-o relative m
o-4 to ovulation Frozen
4-8 (hours?
8-12
semen
Figure 3. Mean number of accessory spermatozoa in zonae of embryos and oocytes following differing times of insemination relative to ovulation using liquid (Experiment 1; n=76) or frozen (Experiment 2; n=SO) semen.
839
Theriogenology
increased rates of embryonic loss during early pregnancy, compared to preovulatory inseminated gilts. Recently it was demonstrated that delayed mating in relation to ovulation decreased mean blastocyst size and estrogen secretion in vitro when blastocysts were collected 288 h post-hCG (2). The decrease in fertilization rates following postovulatory insemination resulted from an increased number of unfertilized ova while the percentage of degenerated embryos remained constant (Experiment 1). Together with the findings of decreased numbers of accessory spermatozoa it can be suggested that the decrease in fertilization rate in delayed inseminated gilts is due to deficiencies in sperm transport as reported earlier by Hunter (5). The more striking decline of pregnancy and fertilization rates after postovulatory insemination with frozen semen compared to liquid semen can be explained by a higher elimination rate in the female genital tract due to cell damage after cryopreservation, resulting in lower sperm numbers at the site of fertilization (11,lS). This was even obvious in the optimal insemination interval close to ovulation, where initial pregnancy and fertilization rates were similar between frozen and liquid semen: the numbers of accessory spermatozoa were ten-times lower using frozen semen compared to liquid semen (Figure 3), although a higher dosage of frozen spermatozoa (5 x log vs 2 x log) was used. In contrast to previous experiments (16,21), no differences in fertilization results were found between frozen semen in round and flat straws. The reason for this is not clear. It might be that in the present study the different intervals of insemination relative to ovulation were the dominating factor influencing the fertilization capacity of frozen semen. In conclusion, the optimal time of insemination using liquid semen is in an interval from 12 to 0 h before ovulation. With frozen semen fertilization results not diiering from those using liquid semen can be achieved when insemination is performed in an interval of approximately 4 h before ovulation. The low tolerance for the optimal timing of AI indicates the importance of tkther studies on sperm transport and ovulation stimulating substances present in seminal plasma (3,4,12,20). In addition, the recently described relationship between the onset of estrus after weaning, the duration of estrus and the time of ovulation (22) offers chances for successful fertiliition using frozen semen in optimally controlled sow herds. REFERENCES 1. Bwanga CO, Hofino PO, Grevle IS, Einarsson S, Rodriguez-Martinez H. In vivo fertilizing capacity of deep frozen boar semen packaged in plastic bags and maxi-straws. J Vet Med A 1991;38:281-286. 2. Ckrdenas II, Pope WF. Effect of time of mating relative to ovulation on morpholgical diversity of swine blastocysts. Biol Reprod 1993;49:1015-1018. 3. Claus R. Physiological role of seminal plasma components in the female reproductive tract. J Reprod Fertil 1990; 40 (Suppl): 117- 13 1. 4. Einarsson S, Viring S. Distribution of frozen-thawed spermatozoa in the reproductive tract of gilts at different time intervals after insemination. J Reprod Fertil 1973;32:117-120. 5. Hunter RHF. The effects of delayed insemination on fertilization and early cleavage in the pig. J Reprod Fertil 1967;13: 133-147. 6. Hunter RHF. Physiological factors influencing ovulation, fertilization, early embryonic development and establishment of pregnancy in pigs. Br Vet J 1977;133:461-470.
840 7. Hunter RHF. Interrelationships
8. 9.
10.
11.
12.
13. 14. 15.
16. 17. 18.
19. 20.
21.
22.
23.
Theriogenology
between spermatozoa, the female reproductive tract, and the egg investments. In: Cole DJA, Foxcroft GR (eds), Control of Pig Reproduction, Buttersworth Scientilic, London, 1982; 49-63. Hunter RI-IF. Oviduct iimction in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991;29:385-39i. Johnson LA Fertility results using frozen boar semen: 1970 to 1985. In: Johnson LA, Larsson K (eds), Deep Freezing of Boar Semen, Uppsala, Sweden, 1985; 199-222. Larsson K. Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation. Intluence of boars and thawing diluents. Acta Vet Stand 1976;17:63-73. Purse1 VG, Schulmann LL, Johnson LA. Distribution and morphology of fresh and frozen thawed sperm in the reproductive tract of gilts after artificial insemination. Biol Reprod 1978;19:69-76. Rath D, Weitze KF, Pena Alfaro CE, Andrade Moura JC. Effects of seminal plasma on the number of accessory sperm cells and fertilization in gilts. Zuchthygiene 1989;24: 123127. Saacke RG. Semen quality in relation to semen preservation. J Dairy Sci 1982;66:26352644. SAS User’s Guide: Statistics SAS Institute, Inc, Cary, 1985. Scheffels W, Biegert W, Leidl W. Die Verteihmg der Spermien im weiblichen Genitale des Schweines nach Insemination von fXschem und tiefgefrorenen Sperma. Zuchthygiene 1971;6:60-64. Simmet C, Gall T, Waberski D, Weitze KF. Fertility of boar semen frozen in flat straws and macrotubes. Reprod Dom Anim 1993;2 (Suppl):87 abstr. Waberski D, Weitze KF, Rath D, Salhnann HP. Wnkung von bovinem Serumalbumin und Zwitterionenpuffer auf fliissigkonservierten Ebersamen. Zuchthygiene 1989;24: 128-133. Waberski D, Weitze KF, Lietmann C, Ltibbert zur Lage W, Bortolozzo FP, Wilhnen T, Petzoldt R The initial fertilizing capacity of longterm-stored liquid boar semen following pre- and postovulatory insemination. Theriogenology 1994;4 1: 1367- 1377. Weitze KF, Habeck 0, Willmen T, Rath D. Detection of ovulation in the sow using transcutaneous sonography. Zuchthygiene 1989; 24140-42. Weitze KF, Lotz JH, Everwand A, Willmen T, Waberski D. Interaction between inseminate, uterine and ovarial fimction in the sow. II. Investigations into the influencing of ovulation by the use of sperm-&e media. Reprod Dom Anim 1990;25: 197-204. Weitze KF, Stampa E, Richter L, Wdhnen T, Waberski D. Fertility of frozen boar semen: influence of packaging, number of inseminations, and seminal plasma. Reprod Dom Anim 1991;l (Suppl):139-142. Weitze KF, Waberski D, Wagner-Rietschel II, Richter L, Kalm E. The onset of heat after weaning, heat duration, and ovulation as major factors in AI timing in sows. Reprod Dom Anim (m press). WestendorfP, Richter L, Treu H. Zur Tiefgefiierung von Ebersperma: Labor- und Besamungsergebnisse mit dem Htilsenberger Pailletten-Verfahren. Dtsch tier&rztl Wochenschr 1975;82,261-267.