Effect of age on quality of fresh and frozen-thawed semen in White Italian ganders

E. Lukaszewicz¹, W. Kruszynski², N. Fujihara³

Asian J Androl 2003 Jun; 5: 89-93             

Keywords: poultry; gander; age; semen; sperm morphology; freezing

Abstract

Aim: To comparatively evaluate the fresh semen quality of 1, 2 and 3-yr-old White Italian ganders (Anser anser L.) and the susceptibility of spermatozoa to freezing-thawing procedure. Methods: Semen was collected by dorso-abdominal massage every 2 days~3 days from three groups of ganders: 1-yr-old (n=11), 2-yr-old (n=7) and 3-yr-old (n=9). In the pooled fresh semen samples, the following parameters were evaluated: the ejaculate volume, the blood or fecal contamination and the motility, concentration and morphology of spermatozoa. Sperm motility and morphology were evaluated in the frozen-thawed semen. Semen diluted with EK extender was frozen in straws in a computerized freezing unit with 6 % dimethyl-formamide to -140 at a rate 60 /min and then transferred into the LN₂ container. Straws with semen were thawed in a water bath at 60 . Results: The ejaculate volume decreased with the age (0.21 mL for 1-yr-old, 0.18 mL for 2-yr-old and 0.14 mL for 3-yr-old ganders); the sperm concentration increased with the age (32710⁶ mL^-1 for 1-yr-old, 43110⁶ mL^-1 for 2-yr-old and 54710⁶ mL^-1 for 3-yr-old ganders); the number of live - normal sperm was significantly (P<0.01) lower in the 1-yr-old than that in the 2- and 3-yr-old ganders (26.61 %, 41.54 and 35.9 %, respectively). The percentage of normal cells survived the freezing-thawing process was 37.7 %, 43.3 % and 40.9 % for 1-, 2- and 3-yr-old ganders, respectively. Conclusion: Freezing and thawing processes more significantly (P<0.01) affected the motility, viability and morphology of spermatozoa in semen of 1-yr-old ganders in comparison with older males.

1 Introduction

Domestic geese are seasonal breeders and among all poultry species, have the lowest reproductive capability, a reflection of poor semen quality and low egg production, fertility and hatchability rates. Geese can live for 20 years or more [1], but their reproductive potential remains on acceptable level only up to the fifth reproductive season. They are usually kept for 3 to 4 seasons in breeding practices, however in the first reproductive season the egg fertilization and hatchability rates are lower than those in the next seasons, despite that the number of egg production during the first five reproductive seasons was similar [2]. Incapability to mate successfully or poor semen quality in young ganders are possible causes of the low reproductive efficiency of 1-yr-old geese flocks mated naturally. Studies conducted to evaluate the semen quality in ganders during the entire reproductive season showed that there were larger semen volume and lower proportion of live-morphologically normal spermatozoa in 1-yr-old males than those in 2-yr-old males [3].

Semen cryopreservation is one of the methods to preserve the genetic potential of domestic and wild birds. It has been demonstrated that semen of wild birds [4, 5] and different poultry species [6-12] could be frozen successfully and that satisfactory fertility level could be obtained after insemination with frozen-thawed semen. Success in freezing semen of a particular poultry species depends on many factors, including the freezing medium (extenders and cryoprotectant), the equilibration time, the freezing rate and the thawing temperature [5, 12]. However, one of the most important factors is the quality of semen subject to freezing [13]. Quality of gander semen depends on the species, breed, feeding, management system, reproductive season and semen collection procedure [3, 5, 14]. Since age also affects the fresh semen quality in ganders, age may also affect the ability of sperm to withstand the freezing-thawing process.

The objective of the present investigation was to study the sperm production and the quality of fresh and frozen-thawed semen in 1-, 2- and 3-yr old White Italian ganders.

2 Materials and methods

This experiment was conducted at the Department of Poultry Breeding, Agricultural University of Wroclaw, Poland. Three groups of White Italian ganders (Anser anser L.) were used as semen donors, including Group 1: 1-yr-old males (n=11), Group 2: 2-yr-old males (n = 7) and Group 3: 3-yr-old males (n = 9). The birds were kept in individual cages under natural light and temperature conditions were fed with 400 g/day to 450 g/day of a commercial ration for breeding geese, which contained 11.1 MJ metabolize energy and 140 g crude protein/kg. In order to maximize the semen quality, the birds were periodically 'bathed' in a water basin, which contributed to feather clearness and thus helped to improve the clearness of the ejaculates.

Semen was collected by dorso-abdominal massage three times a week during the middle of the reproductive season (April) and twice a week at the end of the reproductive season (May). A total of 16 collections were performed for Group 1, and 18 collections each for Groups 2 and 3. During the collections, a particular care was taken to keep the same regime, i.e. environment, time of collection, staff members and massage method in order to avoid excessive stress and possible deleterious effects. Semen collection was completed within 20 min to 25 min and the freezing procedure started within 30 min from the beginning of the collections.

Ejaculate colour was evaluated by visual inspection to assess blood or faecal contamination. Clean ejaculates were transferred into conical glass tubes to form a pooled semen sample for each group. Ejaculate volume, and sperm concentration, motility and morphology were determined in pooled fresh semen samples. Total pooled ejaculate volume was determined with a pipette and the average ejaculate volume was calculated by dividing the total pooled volume by the number of birds collected on the particular day of sampling. Sperm concentration was determined by the hemocytometer method. For better evaluation of the fresh semen quality, especially with the respect to the number of spermatozoa potentially capable of fertilisation, the semen quality factor (SQF), calculated according to following equation, was employed:

SQF = [Sperm concentration per mLejaculate volume (mL)live-normal spermatozoa (%)]/ 100 %

Sperm motility was assessed with the Blom's chamber, which allows observing sperm in 3 layers with different thickness (5 mm, 50 mm and 350 mm). Collective sperm motility was determined at the 350 mm-thick layer and graded in a 5 point's scale (Point 5: 80 %~100 %, Point 4: 60 %~80 %; Point 3: 40 %~60 %; Point 2: 20%~40 %; Point 1: below 20 %). Individual sperm motility was determined at the 5 m-thick layer and expressed as a percentage of motile sperm ranging from 10 % to 100 % [15].

Sperm morphology was determined in eosin/nigrosin-stained smears evaluated under a 1,250 light microscope (Jenaval, Carl Zeiss, Jena, Germany). For each smear, 300 spermatozoa were evaluated and categorised into 7 classes. Classes 1 to 6 consisted of live (unstained) cells: 1-morphologically normal spermatozoa (typical spindle-shaped head and well-marked acrosome); 2-macro-cephalic spermatozoa; 3-bent-neck spermatozoa; 4- midpiece deformed spermatozoa (swelling, ragged or lack of midpiece); 5-spermatids (immature cells with a spherical head and tail only); 6-spermatozoa with other defects; 7-dead spermatozoa (partially or completely stained by eosin). Spermatids were taken into account only in fresh semen samples as an indication of testicular function [16]. The results of the morphological examination were expressed as the proportion of the particular class of spermatozoa.

Pooled semen samples were frozen in straws according to the method described for the White Italian gander [9, 17]. Briefly, semen samples were diluted with EK extender, equilibrated for 15 minutes at 4 and mixed with dimethyl-formamide (DMF; SERVA, Heidelberg, Germany) to a final concentration of 6 % (v/v). Loaded straws were frozen to -140 at a rate of 60 /min with a programmable freezing unit (Minidigitcoll 1400; IMV, LAigle, France) and then plunged into a liquid nitrogen container. Frozen straws were thawed in the water bath at 60 for a few seconds. Sperm motility and morphology were determined in frozen-thawed samples in the same way.

2.4 Statistical analysis

The results of sperm morphology evaluation were transformed by arc sin?/FONT>x prior to analysis. The effects of age on ejaculate volume, sperm concentration, motility and morphology were determined by ANOVA in fresh semen, while ANCOVA was used to determine the effect of age on sperm motility and morphology in frozen-thawed samples. Duncan's multiple range test was used to locate significant differences (SAS system, General Linear Models Procedure; SAS Institute, Cary, USA). Correlation between traits evaluated in fresh and frozen-thawed semen were determined using SPSS/PC (Arizona State University, USA). In order to evaluate the effect of freezing on sperm morphology, relative changes (RCH) between fresh and frozen-thawed samples were calculated considering the percentages observed in fresh semen as 100 %.

3 Results

Ejaculate volume decreased (P=0.01) with the age, while sperm concentration increased (P<0.01; Table 1); the correlation between ejaculate volume and sperm concentration was negative (r=-0.4; P=0.01). There was no well-marked difference in sperm motility among groups; the collective motility was rather poor and never exceeded 3 points, while individual sperm progressive motility ranged from 50 % to 60 %.

Table 1. Fresh semen characteristics of 1-, 2- and 3-yr-old White Italian ganders (meanSD). ^aP<0.05 (Group 1 vs. Group 2 and 3), ^bP< 0.05 (Group 1 vs. Group 2 and 3, Group 2 vs. Group 3), ^cP<0.05 (Group 1 vs. Group 2), ^dP<0.05 (Group 1 vs. Group 3).

The percentage of live - morphologically normal cells in the fresh semen of 1-yr-old ganders was significantly lower (P<0.01) in comparison with older ganders. It varied from 18.7 % to 41.0 %, while in the semen of 2- and 3-yr-old males, from 26.0 % to 53.3 % and from 27.3 % to 44.0 %, respectively. The percentages of dead sperm and sperm with bent-neck, midpiece deformities and other defects were greater (P=0.05) in 1-yr- old ganders than those in 2-yr-old ganders, while 3-yr-old ganders were intermediate. There was a high (P< 0.01) and negative correlation between the number of these forms of spermatozoa and live-morphologically normal ones in the fresh semen (Table 3). The semen quality in the youngest males, expressed as SQF, was also significantly lower (P<0.01) than that in the older ganders (Table1).

The freezing and thawing negatively affected the sperm motility and morphology. Collective sperm motility was slower in comparison with the fresh semen and the individual motility was approximately 40 %, but 10 % cells revealed abnormal, pulsating movement.

Depending on the age of ganders, the number of live - normal sperm decreased by 17.2 % to 23.6 % in comparison with the fresh semen (Table 2). The number of these spermatozoa varied from 6.7 % to 14.7 % in thawed semen of 1-yr-old males; 11.3 % to 23.0 % in the 2-yr-old and from 8.3 % to 23.0 % in the 3-yr-old ganders. The percentage of live-morphologically normal cells in the frozen-thawed semen was positively correlated with the total number of live spermatozoa in the fresh semen (P<0.01; Table 3). Taking into account the RCH values (changes in relation to the fresh semen), the average number of spermatozoa that survived freezing-thawing process and remained morphologically intact was the highest (43.2 %) for 2-yr-old ganders (Table 2). There were small changes in the percentage of live-defective sperm. In some cases the percentage of defective sperm decreased in the frozen-thawed samples, which could be explained by the fact that defective sperm did not survive the freezing-thawing process thus the percentage of dead cells in frozen-thawed samples increased. The number of deformed sperm in the fresh semen was negatively correlated with the live-normal cells in the thawed semen, but only the correlation between bent-neck and live - normal spermatozoa was significant (P< 0.01; Table 3).

Table 2. Morphology of frozen-thawed semen and relative changes between fresh and frozen-thawed semen of 1, 2 and 3-yr-old White Italian ganders. Relative change: % spermatozoa survived the freezing process (fresh semen: 100 %). ^aP<0.05 (Group 1 vs. Group 2 and 3). ^bP<0.05 (Group 1 vs. Group 2 and 3, Group 2 vs. Group 3). ^cP<0.05 (Group 1 vs. Group 2).

Table 3. Correlation coefficients among morphological classes of spermatozoa estimated in fresh and frozen-thawed semen of 1-, 2- and 3-yr-old White Italian ganders. TL- total live; LN- morphologically normal & live, MCPH- macrocephalic, BN- bent-neck, MDF- midpiece defects, OTH- other defects, Dead- dead spermatozoa. ^cP0.01.

4 Discussion

The results of the present study suggested that low sperm production and semen quality of 1-yr-old ganders may be responsible for the low reproductive capability, i.e., low egg fertility and hatchability rates, observed in the first reproductive cycle of goose flocks mated naturally [2]. The semen quality expressed as SQF increased with ganders' age.

It should be emphasised that relatively low number of live-morphologically intact spermatozoa was observed generally in semen of ganders derived from Anser anser L. species [3, 9, 16, 17]. In the present experiment, the percentage of total live sperm was high, varying from 91 % to 95 % irrespective of age, but the percentage of live-morphologically normal sperm was not greater than 27 % to 41%; there were high percentages of macro-cephalic sperm and sperm with bent-neck. Studies had demonstrated that the number of bent-neck sperm in fresh and frozen-thawed semen was negatively correlated with the fertility in fowls [18] and in fresh semen with White Italian geese [16]. Johnson [19] and Chelmonska [16] concluded that a high number of defective sperm and spermatids observed in the gander semen indicate that the process of spermatogenesis was limited in this species.

The positive correlation between the percentage of live-normal sperm in fresh and frozen-thawed semen in the present experiment (r=0.5043), as well as in other studies [13] indicated that the quality of fresh semen is of fundamental importance in the successful cryopreservation of poultry semen. In comparison with older males, 1-yr-old ganders produced the worst quality semen with spermatozoa most vulnerable to the freezing process.

Further experiments should be carried out in order to explain which of the fresh semen characteristic influences the susceptibility of the spermatozoa to freezing-thawing process.

The authors would like to express cordially thanks to Mrs. Anna Jerysz and Mr. Tadeusz Wozniak for a great help and assistance during the experiment.

References

[1] Bieliska K. History of the domestic goose. Poultry Production (in Polish) 1979; 6: 3-5.
[2] Rosiski A, Bieliski K, Bieliska K. The results of geese reproduction in the same flock during subsequent three years. Annals Animal Breeding Sciences (in Polish) 1986; 13: 127-32.
[3] Lukaszewicz E, Furuta H, Xi YM, Fujihara N. Comparative study on semen quality of one- and two-year-old ganders during the entire reproductive season. Asian J Androl 2000; 2: 139-42.
[4] Gee GF, Sexton TJ. Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia). Zoo Biology 1990; 9: 361-71.
[5] Blanco JM, Gee G, Wildt DE, Donoghue AM. Species variation in osmotic cryoprotectant and cooling rate tolerance in poultry, eagle and peregrine falcon spermatozoa. Biol Reprod 2000; 63: 1164-71.
[6] Bakst MR. Preservation of avian cells. In: Crawford RD, editor. Poultry Breeding and Genetics. Oxford, New York, Tokio: Elsevier Amsterdam; 1990. p 91-108.
[7] Fujihara N, Ohboshi S. Simple and rapid cryopreservation of rooster spermatozoa. Low Temperature Medicine1991; 17: 128-31.
[8] Lake PE, Ravie O, McAdam J. Preservation of fowl semen in LN2 application to breeding programmes. Br Poult Sci 1981; 22: 71-7.
[9] Lukaszewicz E. An effective method for freezing White Italian gander semen. Theriogenology 2002; 58: 19-27.
[10] Tselutin K, Narubina L, Mavrodina T, Tur B. Cryopreservation of poultry semen. Br Poult Sci 1995; 36: 805-11.
[11] Tai JJ, Chen JC, Wu KC, Wang SD, Tai C. Cryopreservation of gander semen. Br Poult Sci 2001; 42: 384-8.
[12] Fujihara N, Buckland RB. The effect of different freezing rates on fertilizing ability of frozen-thawed chicken spermatozoa. Jpn J Anim Reprod 1987; 33: 11-4.
[13] Van Voorst A, Leenstra FR. Fertility rate of daily collected and cryopreserved fowl semen. Poultry Sci 1995; 74: 136-40.
[14] Pawluczuk B. Grunder AA. Comparison of three methods of collecting semen from ganders. Poultry Sci 1989; 68: 1714-7.
[15] Bielaski W. Animal reproduction. PWRiL, editor (in Polish). Warszawa; 1972. p 395-401.
[16] Chelmonska B. Artificial insemination in geese. (in Polish). Med Wet 1967; 7: 432-6.
[17] Lukaszewicz e. Effects of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa. Theriogenology 2001; 55: 1819-29.
[18] Maeda T, Terada T, Tsutsumi Y. Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing. Br Poult Sci 1986; 27: 695-702.
[19] Johnson AS. Artificial insemination and duration of fertility of geese. Poultry Sci 1954; 33 : 638-40.

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Correspondence to: Prof. N. Fujihara, Fukuoka Women's Junior College 6-16-4 Wakagidai, Fukuma, Fukuoka 811-3221, Japan.
Tel/fax: +81-940-435 972
E-mail: nfpantok@proof.ocn.ne.jp
Received 2002-09-23   Accepted 2003-04-21