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Semen Quality Factor as an Indicator of Fertilizing Ability for Geese
Research Note Semen Quality Factor as an Indicator of Fertilizing Ability for Geese S. J. Liu,*† J. X. Zheng,* and N. Yang*1 *Department of Animal Genetics and Breeding, College of Animal Science and Technology & State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100094, China; and †College of Animal Science and Technology, HLJ August First Land Reclamation University, Daqing 163319, China
Key words: goose, semen quality, fertility, testosterone 2008 Poultry Science 87:155–159 doi:10.3382/ps.2007-00300
to fertilize a succession of ova. Only morphologically normal spermatozoa are capable of ascending through the vagina of the hen to the region where the sperm storage tubules are located (Bakst et al., 1994). Traditional poultry semen evaluation methods used in the industry for selection of semen donors either have not been feasible to perform on individuals or have not been predictive of fertility (Donoghue, 1999). Sperm mobility is a primary determinant of fertility in domestic fowl (Donoghue et al., 1998, 1999; Froman and Feltmann, 1998, 2000; Froman et al., 1997, 1999, 2003), and the Accudenz method is very accurate in predicting fertility. Sperm mobility is a function of the product of motile concentration and the proportion of motile sperm with a straight line velocity >30 m/s (Froman et al., 2003). An alternative approach for evaluating the reproductive potential of male breeders is to measure the sperm quality index (SQI; McDaniel et al., 1998; Parker et al., 2000, 2002). The sperm quality analyzer (e.g., AviMate Sperm Quality Analyzer) was used to obtain an SQI score, which provides an estimate of overall sperm quality and quantity in roosters (McDaniel et al., 1998; Parker et al., 2000, 2002). Sperm concentration, viability, and motility all contribute to the SQI. The sperm mobility assay is moderately time-consuming and requires a modicum of equipment at 41°C for 5 min, which leads to certain difficulties in
INTRODUCTION Unlike most domestic birds, geese have a relatively short reproductive period and poor egg production, and the ganders produce a small volume of ejaculate with a low spermatozoa concentration and a low number of live normal spermatozoa (Łukaszewicz, 2000). Goose production has been developing in recent years, but the low reproductive efficiency has hindered its continued expansion. Semen quality is an important factor affecting fertility. Previous studies have demonstrated significant differences among ganders (Johnson, 1954; Pawluczuk and Grunder, 1989; Łukaszewicz, 2002; Łukaszewicz and Kruszynski, 2003), and have shown the necessity of male selection based on semen fertilizing ability (McDaniel, 1995; Donoghue, 1999; Mellor, 2001). Sustained fertility in the avian female depends on its ability to store adequate viable spermatozoa in sperm storage tubules and to supply the infundibulum with sufficient numbers of sperm
©2008 Poultry Science Association Inc. Received July 20, 2007. Accepted September 18, 2007. 1 Corresponding author: [email protected]
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fertility were evaluated. Testosterone and estradiol levels in seminal plasma were determined by competitive immunoassays. The SQF values of the 4 diluted semen groups were 19.3, 11.3, 9.5, and 13.1, and the corresponding fertility values, determined by candling, were 83.9, 65.6, 56.8, and 74.3%, respectively. The correlation coefficient between SQF and fertility was 0.985 (P < 0.01), indicating that the SQF can be used as a good indicator of fertility for geese. The SQF values correlated well with the ratio of testosterone to estradiol in seminal plasma (r = 0.48, P < 0.05), which could also be a diagnostic tool for evaluating the reproductive potential of individual ganders.
ABSTRACT The semen quality factor (SQF) takes into account 3 semen parameters as a means of predicting semen fertilizing ability: semen volume, semen concentration, and the percentage of live and morphologically normal spermatozoa. The objective of this study was to test the suitability of the SQF as an indicator of fertility for geese. Twenty-one ganders were divided into 3 groups based on their individual SQF values, and semen was pooled within each group. A fourth semen group was generated by diluting semen from the highest SQF group with 2 vol of diluent. Pooled semen was inseminated into 30 geese per treatment. Relationships between SQF and
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MATERIAŁ AND METHODS Birds Twenty-one 2-yr-old Yangzhou goose ganders (Anser cygnoides) were kept in individual pens under natural lighting and temperature conditions, with access to a pool of water. One hundred twenty 2-yr-old female geese from a cross of 2 breeds (Lijia and Zi geese, A. cygnoides) were randomly divided into 4 groups of 30 individuals each and kept in separate open-air pens. Pools of water were also made available to the females. All birds were fed 250 to 300 g/d of a commercial ration for goose breeding containing 10.80 MJ of ME and 140 g of CP/kg.
Semen Collection and Determination of Semen Quality The ganders used in this study were selected on the basis of a positive reaction to dorsa-abdominal massage resulting in semen ejaculation. Semen was collected 3 times a week during the middle of the reproductive season (February). Within 30 min after collection, ejaculate volume, motility, concentration, and the morphology of spermatozoa were evaluated. The morphology of spermatozoa was evaluated after nigrosin-eosin staining (Bakst and Cecil, 1997). In each case, 300 spermatozoa per slide (1,600× magnification) were evaluated. Sperm concentration was measured by a hemocytometer, and motility was estimated by using the hanging drop method at 400×
magnification (Bakst and Cecil, 1997). The pH was determined with an EcoScan 5 pH meter (Eutech Instruments Pte Ltd., Singapore), and the volume of semen was measured by a semen collection cup to the minimum 10 L. Semen quality factor values were calculated according to the following equation (Łukaszewicz and Kruszynski, 2003): SQF = ejaculate semen volume (for individual gander evaluation; mL) or insemination dose (for diluted pooled semen evaluation; mL) × sperm concentration (×106/mL) × live and morphologically normal spermatozoa (%).
Semen Treatment Part of the freshly collected semen from each gander was used in determining semen quality, whereas the remainder was centrifuged at 800 × g for 10 min to collect the seminal plasma. Samples of seminal plasma were stored at −20°C until assayed.
Fertilizing Ability Test Based on the SQF of the last 3 evaluations, the 21 ganders were divided into 3 groups: high (H) SQF (n = 4, range from 117.8 to 264.3), medium (M) SQF (n = 7, range from 75.5 to 117.0), and low (L) SQF (n = 10, range from 20.2 to 68.7). The consideration of sufficient insemination volume needed for 30 geese resulted in uneven sample sizes for each group. Pooled semen of the H SQF group was diluted either 1:1 or 1:2 (semen:diluent) with Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen Corp., Carlsbad, CA). The remaining 2 SQF semen groups were diluted 1:1. In the end, there were 4 groups of diluted semen, namely, H(1:1), M(1:1), L(1:1), and H(1:2). The 4 groups of female geese were randomly inseminated with the 4 groups of diluted semen within 30 min of semen collection. Each goose was inseminated with 0.1 mL of diluted semen. The SQF for pooled semen was also calculated for each group. Two inseminations were performed within an interval of 5 d. Hatching eggs were collected daily from d 2 after the first insemination to d 6 after the last insemination. The eggs collected in 11 d were set in an automatic incubator and incubated at 37.8°C. Fertility was determined by candling on d 10 of incubation.
Hormone Measurements Seminal plasma testosterone and estradiol concentrations were determined by competitive immunoassay with an Immulite system (Diagnostic Products Corp., Los Angeles, CA). The intra- and interassay CV were 7.1 and 6.3% for estradiol, and 7.5 and 7.7% for testosterone, respectively. The sensitivities of the assays were 55 pmol/ L for estradiol and 0.5 nmol/L for testosterone. The im-
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field tests. The SQI is measured in 20 s at room temperature, but a sperm mobility phenotype is not determined by the SQI (Froman et al., 2003). Because of significant differences in the quantity and quality of ejaculates among individual ganders, the semen quality factor (SQF) has been proposed as a predictor of gander semen fertilizing ability (Łukaszewicz and Kruszynski, 2003). The SQF is a composite of 3 important semen traits: semen volume, semen concentration, and the percentage of live and morphologically normal spermatozoa. High-quality semen relies on normal spermatogenesis. It is becoming increasingly evident that estrogen, in addition to testosterone, plays a role in the development and function of the testis and male reproductive tract (Rivas et al., 2002; Akingbemi, 2005). The hormone level in seminal plasma is a direct reflection of male testicular endocrine activity. The relationships between semen quality and concentration of testosterone in avian seminal plasma have been discussed (Zeman et al., 1986; Cecil and Bakst, 1988). Although many sperm quality indices are used in evaluating avian semen quality, a method that accurately predicts fertility and is feasible to apply under field conditions is lacking. In the current study, the suitability of SQF in evaluating gander fertility was explored, and the roles of sex hormones in gander reproductive function were examined.
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RESEARCH NOTE Table 1. Descriptive statistics of semen quality parameters Item Mean ± SEM Range CV (%)
1
Volume (mL)
Concentration (×106/mL)
Live and normal morphology (%)
Motility (%)
pH
SQF2
0.41 ± 0.05 0.18–0.90 51.21
701 ± 82.3 230–2020 53.49
33.9 ± 2.3 28.0–51.7 30.38
54.2 ± 1.8 40.1–67.0 15.68
7.10 ± 0.02 7.02–7.21 1.41
84.0 ± 11.8 20.2–264.3 64.52
1
n = 21. Semen quality factor (SQF) = ejaculate volume (mL) × sperm concentration (×106/mL) × live and morphologically normal spermatozoa (%). 2
munoassays were validated for measurement of hormones in semen by addition of the following standards: 17β estradiol, 1:230 pmol/L, and testosterone, 1.0 nmol/L.
The levels of sex hormones were logarithmically transformed to obtain a normal distribution. The ratio of testosterone:estradiol was calculated and logarithmically transformed. Differences in the levels of sex hormones and the SQF among the 3 groups were analyzed by ANOVA, and the significance was determined by Duncan’s multiple-range tests (SAS Institute, 2001). The correlations between sex hormones and SQF were calculated with a Pearson correlation, and Spearman’s rank correlation was used to evaluate the relationship between SQF and fertility.
RESULTS Semen Quality and Fertility The SQF values of individual ganders varied from 20.2 to 264.3. Means for ejaculate volume, sperm concentration, live and morphologically normal spermatozoa, motility, pH, and SQF were 0.41 mL, 701 × 106/mL, 33.9%, 54.2%, 7.10, and 84.0, respectively (Table 1). Very large variations among ganders were found for all semen parameters except for pH. The CV for ejaculate volume, sperm concentration, live and morphologically normal spermatozoa, and motility were 51.21, 53.49, 30.38, and 15.68%, respectively, resulting in a CV as high as 64.52% for SQF. There were significant differences among the 3 gander groups (P < 0.05); the values were 155.2 ± 36.3, 86.7 ± 8.1, and 54.1 ± 7.3, respectively. The SQF values based on pooled semen were 19.3, 11.3, 9.5, and 13.1 for the H(1:1), M(1:1), L(1:1), and H(1:2) semen groups, respectively (Table 2). The corresponding fertility values for the different groups of geese were 83.9, 65.6, 56.8, and 74.3% for the respective semen groups, which showed the same trend as the changes in SQF. The Spearman rank correlation coefficient between SQF and fertility was 0.985 (P < 0.01).
Testosterone and Estradiol Concentrations in the 3 Gander Groups No significant difference among the different groups was observed for the levels of testosterone and estradiol
Correlation Between SQF and Sex Hormones For all 21 samples, SQF was negatively correlated with estradiol concentration (r = −0.68, P < 0.05) and positively correlated with the ratio of testosterone:estradiol (r = 0.48, P < 0.05).
DISCUSSION Turkey production in the United States relies mainly on artificial insemination, and artificial insemination is used nearly 100% in turkey breeding (Holsberger et al., 1998; Donoghue et al., 1999). Artificial insemination in goose breeding, although not used as routinely as in turkey and chicken breeding, is a very important technique that can help to eliminate ganders with poor semen quality so as to necessitate the selection of males that are
Table 2. Effects of different semen quality factor (SQF) values on geese fertility Group1
SQF2
Eggs set3 (n)
Fertile eggs (n)
Fertility (%)
H(1:1) M(1:1) L(1:1) H(1:2)
19.3 11.3 9.5 13.1
124 96 111 109
104 63 63 81
83.9 65.6 56.8 74.3
1 (1:1) H , H(1:2) = high SQF group, in which semen was diluted in 1:1 or 1:2 with Dulbecco’s modified Eagle’s medium (DMEM), respectively; M(1:1) = medium SQF group, in which semen was diluted in 1:1 with DMEM; L(1:1) = low SQF group, in which semen was diluted in 1:1 with DMEM. 2 SQF = semen quality factor for 1 dose (0.1 mL) of pooled semen in each group measured after insemination. 3 Data for eggs collected from d 2 after the first insemination to d 6 after the last insemination.
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Statistical Analysis
in seminal plasma (Figure 1). The levels of testosterone varied from 9.3 ± 1.3 nmol/L (the H SQF group) to 6.3 ± 1.9 nmol/L (the M group) and 6.4 ± 1.2 nmol/L (the L SQF group), and the corresponding values of estradiol increased from 683 ± 119 pmol/L to 740 ± 51 pmol/L and 905 ± 130 pmol/L. The ratios of testosterone to estradiol in the 3 groups were 15.5 ± 3.3, 8.6 ± 2.2, and 8.0 ± 1.4, respectively. The ratio of testosterone to estradiol in the H group was higher than that of the L group (P < 0.05). There was no significant difference between the M group and the L group or between the H group and the M group.
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producing the highest quality semen, or to overcome the difficulties of natural mating caused by the large difference in body size between males and females as well as the low libido of ganders when crossing with females from different breeds. Fertility results for artificial insemination of geese using fresh semen vary widely in the literature. Behr and Jochims (1992) recognized that satisfactory fertility can be achieved if the quantity of insemination does not decrease below 5 million per goose, but Malis (1998) obtained only 65% fertile eggs collected from weekly goose insemination with 10 million fresh spermatozoa. The SQF was used successfully as a means to evaluate semen quality in the current experiment, highlighting the differences among semen fertilizing ability. Very large variation in ejaculate volume was observed in ganders, indicating the importance of including ejaculate volume in evaluating the reproductive potential of ganders. The SQF, which takes into account the semen volume, shows an advantage over the SQI and over mobility in screening better ganders for reproduction. The current study showed a significant positive correlation between SQF and fertility, implying that the SQF could best be used as an indicator of fertility in goose artificial insemination. The evaluation of sperm morphology does not require sophisticated facilities and equipment and can be performed under field conditions. Evaluations repeated by the same person avoid operator-dependent errors (Łukaszewicz and Kruszynski, 2003). On the basis of the specific semen quality parameters in a pooled semen sample, the operator can predict the number of insemination doses needed to achieve satisfactory fertility. Several steps of spermatogenesis are under the control of estrogen via genomic and nongenomic pathways (Carreau et al., 2006). The role of estrogen in male reproduction is, in part, related to the ratio of androgens:estrogens
ACKNOWLEDGMENTS We would like to thank Kangning Zhang from the Changzhou Siji Goose Company of Jiangsu Province, China, for his assistance in the experiment. The current research was supported in part by the National Basic Research Program of China (2006CB102102).
REFERENCES Akingbemi, B. T. 2005. Estrogen regulation of testicular function. Reprod. Biol. Endocrinol. 3:1–13. Bakst, M. R., and H. C. Cecil. 1997. Techniques for semen evaluation, semen storage, and fertility determination. Poult. Sci. Assoc., Savoy, IL. Bakst, M. R., G. Wishart, and J. P. Brillard. 1994. Oviductal spermatozoa selection, transport, and storage in poultry. Poult. Sci. Rev. 5:117–143. Behr, K.-P., and U. Jochims. 1992. Artificial insemination of geese under field conditions. Page 684 in Proc. XIX World Poult. Congr., Amsterdam, the Netherlands. Vol. 1. World’s Poult. Sci. Assoc., Netherlands Branch, Wageningen. Carreau, S. C., C. Delalande, D. Silandre, S. Bourguiba, and S. Lambard. 2006. Aromatase and estrogen receptors in male reproduction. Mol. Cell. Endocrinol. 246:65–68. Cecil, H. C., and M. R. Bakst. 1988. Testosterone concentrations in blood and seminal plasma of turkeys classified as high or low semen producers. Poult. Sci. 67:1461–1464. Donoghue, A. M. 1999. Prospective approaches to avoid flock fertility problem: Predictive assessment of sperm function traits in poultry. Poult. Sci. 78:437–443. Donoghue, A. M., D. R. Holsberger, D. P. Evenson, and D. P. Froman. 1998. Semen donor selection by in vitro sperm mobility increases fertility and semen storage in the turkey hen. J. Androl. 19:295–301. Donoghue, A. M., T. S. Sonstegard, L. M. King, E. J. Smith, and D. W. Burt. 1999. Turkey sperm mobility influences paternity in the context of competitive fertilization. Biol. Reprod. 61:422–427. Froman, D. P., E. R. Bowling, and J. L. Wilson. 2003. Sperm mobility phenotype not determined by sperm quality index. Poult. Sci. 82:496–502. Froman, D. P., and A. J. Feltmann. 1998. Sperm mobility: A quantitative trait of the domestic fowl (Gallus domesticus). Biol. Reprod. 58:379–384.
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Figure 1. Comparison of the levels of sex hormones and the ratios of testosterone:estradiol in different semen quality factor (SQF) groups. Values are means. Vertical lines represent the SEM. a,bValues without a common letter are different (P < 0.05). Data for the high SQF group (n = 4) are shown by black bars, data for the medium SQF group (n = 7) are shown by diagonally striped bars, and data for the low SQF group (n = 10) are shown by open bars.
(Lambard et al., 2005), and the ratio between androgen and estrogen, rather than their absolute levels, is important for spermatogenesis (Sharpe, 1998; Rivas et al., 2002; Akingbemi, 2005). In the current study, we observed a corresponding reduction in the ratio of testosterone:estradiol in the L SQF group compared with the H SQF group. The SQF was moderately correlated with the ratio of testosterone:estradiol (r = 0.48, P < 0.05), suggesting that a balance between testosterone and estradiol in seminal plasma is important for gander spermatogenesis. This implies that the ratio between testosterone and estradiol may be a diagnostic tool for evaluating the reproductive potential of individual ganders. In conclusion, the SQF is not only a parameter for evaluating the reproductive potential of individual ganders, but is also a good indicator of goose fertilizing ability. A trained worker can easily use this technique in artificial insemination to improve the reproductive efficiency of goose production.
RESEARCH NOTE
McDaniel, G. R. 1995. Managing broiler breeds for maximum fertility. World’s Poult. Sci. J. 9:25–27. McDaniel, C. D., J. L. Hannah, H. M. Parker, T. W. Smith, C. D. Schultz, and C. D. Zumwalt. 1998. Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index. Poult. Sci. 77:888–893. Mellor, S. 2001. Selecting males by sperm quality. World’s Poult. Sci. J. 3:32–34. Parker, H. M., J. B. Yeatman, C. D. Schultz, C. D. Zumwalt, and C. D. McDaniel. 2000. Use of a sperm analyzer for evaluating broiler breeder males. 2. Selection of young broiler breeder roosters for the sperm quality index increases fertile egg production. Poult. Sci. 79:771–777. Parker, H. M., A. G. Karaca, J. B. Yeatman, L. R. Frank, and C. D. McDaniel. 2002. Fertility of broiler breeders following categorization by the Optibreed sperm quality index when hens are inseminated with a constant number of sperm. Poult. Sci. 81:239–245. Pawluczuk, B., and A. A. Grunder. 1989. Comparison of three methods of collecting semen from ganders. Poult. Sci. 68:1714–1717. Rivas, A., J. S. Fisher, C. McKinnell, N. Atanassova, and R. M. Sharpe. 2002. Induction of reproductive tract developmental abnormalities in the male rat by lowering androgen production or action in combination with a low dose of diethylstilbestrol: Evidence for importance of the androgen-estrogen balance. Endocrinology 143:4797–4808. SAS Institute. 2001. Version 8.2. SAS Inst. Inc., Cary, NC. Sharpe, R. M. 1998. The roles of oestrogen in the male. Trends Endocrinol. Metab. 9:371–377. Zeman, M., J. Kosutzky, and E. Bobakova. 1986. Testosterone concentration in the seminal plasma of cocks. Br. Pout. Sci. 27:261–266.
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Froman, D. P., and A. J. Feltmann. 2000. Sperm mobility: Phenotype in roosters (Gallus domesticus) determined by concentration of motile sperm and straight line velocity. Biol. Reprod. 62:303–309. Froman, D. P., A. J. Feltmann, and D. J. McLean. 1997. Increased fecundity resulting from semen donor selection based upon in vitro sperm mobility. Poult. Sci. 76:73–77. Froman, D. P., A. J. Feltmann, M. L. Rhoads, and J. D. Kirby. 1999. Sperm mobility: A primary determinant of fertility in the domestic fowl (Gallus domesticus). Biol. Reprod. 61:400– 405. Holsberger, D. R., A. M. Donoghue, D. P. Froman, and M. A. Ottinger. 1998. Assessment of ejaculate quality and sperm characteristics in turkeys: Sperm mobility phenotype is independent of time. Poult. Sci. 77:1711–1717. Johnson, A. S. 1954. Artificial insemination and the duration of fertility of geese. Poult. Sci. 33:638–640. Lambard, S., D. Silandre, C. Delalande, I. D. Galeraud, S. Bourguiba, and S. Carreau. 2005. Aromatase in testis: Expression and role in male reproduction. J. Steroid Biochem. Mol. Biol. 95:63–69. Łukaszewicz, E. 2000. Effect of semen filtration and dilution rate on morphology and fertility of frozen gander spermatozoa. Theriogenology 55:1819–1829. Łukaszewicz, E. 2002. An effective method for freezing White Italian gander semen. Theriogenology 58:19–27. Łukaszewicz, E., and W. Kruszynski. 2003. Evaluation of fresh and frozen-thawed semen of individual ganders by assessment of spermatozoa motility and morphology. Theriogenology 59:1627–1640. Malis, P. Z. 1998. The optimal use of goose sperm for fertility. Pages 820–824 in Proc. 10th Eur. Poult. Conf., Jerusalem, Israel. World’s Poult. Sci. Assoc., Israel Branch, Rehovot.
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Key words: goose, semen quality, fertility, testosterone 2008 Poultry Science 87:155–159 doi:10.3382/ps.2007-00300
to fertilize a succession of ova. Only morphologically normal spermatozoa are capable of ascending through the vagina of the hen to the region where the sperm storage tubules are located (Bakst et al., 1994). Traditional poultry semen evaluation methods used in the industry for selection of semen donors either have not been feasible to perform on individuals or have not been predictive of fertility (Donoghue, 1999). Sperm mobility is a primary determinant of fertility in domestic fowl (Donoghue et al., 1998, 1999; Froman and Feltmann, 1998, 2000; Froman et al., 1997, 1999, 2003), and the Accudenz method is very accurate in predicting fertility. Sperm mobility is a function of the product of motile concentration and the proportion of motile sperm with a straight line velocity >30 m/s (Froman et al., 2003). An alternative approach for evaluating the reproductive potential of male breeders is to measure the sperm quality index (SQI; McDaniel et al., 1998; Parker et al., 2000, 2002). The sperm quality analyzer (e.g., AviMate Sperm Quality Analyzer) was used to obtain an SQI score, which provides an estimate of overall sperm quality and quantity in roosters (McDaniel et al., 1998; Parker et al., 2000, 2002). Sperm concentration, viability, and motility all contribute to the SQI. The sperm mobility assay is moderately time-consuming and requires a modicum of equipment at 41°C for 5 min, which leads to certain difficulties in
INTRODUCTION Unlike most domestic birds, geese have a relatively short reproductive period and poor egg production, and the ganders produce a small volume of ejaculate with a low spermatozoa concentration and a low number of live normal spermatozoa (Łukaszewicz, 2000). Goose production has been developing in recent years, but the low reproductive efficiency has hindered its continued expansion. Semen quality is an important factor affecting fertility. Previous studies have demonstrated significant differences among ganders (Johnson, 1954; Pawluczuk and Grunder, 1989; Łukaszewicz, 2002; Łukaszewicz and Kruszynski, 2003), and have shown the necessity of male selection based on semen fertilizing ability (McDaniel, 1995; Donoghue, 1999; Mellor, 2001). Sustained fertility in the avian female depends on its ability to store adequate viable spermatozoa in sperm storage tubules and to supply the infundibulum with sufficient numbers of sperm
©2008 Poultry Science Association Inc. Received July 20, 2007. Accepted September 18, 2007. 1 Corresponding author: [email protected]
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Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 11, 2014
fertility were evaluated. Testosterone and estradiol levels in seminal plasma were determined by competitive immunoassays. The SQF values of the 4 diluted semen groups were 19.3, 11.3, 9.5, and 13.1, and the corresponding fertility values, determined by candling, were 83.9, 65.6, 56.8, and 74.3%, respectively. The correlation coefficient between SQF and fertility was 0.985 (P < 0.01), indicating that the SQF can be used as a good indicator of fertility for geese. The SQF values correlated well with the ratio of testosterone to estradiol in seminal plasma (r = 0.48, P < 0.05), which could also be a diagnostic tool for evaluating the reproductive potential of individual ganders.
ABSTRACT The semen quality factor (SQF) takes into account 3 semen parameters as a means of predicting semen fertilizing ability: semen volume, semen concentration, and the percentage of live and morphologically normal spermatozoa. The objective of this study was to test the suitability of the SQF as an indicator of fertility for geese. Twenty-one ganders were divided into 3 groups based on their individual SQF values, and semen was pooled within each group. A fourth semen group was generated by diluting semen from the highest SQF group with 2 vol of diluent. Pooled semen was inseminated into 30 geese per treatment. Relationships between SQF and
156
LIU ET AL.
MATERIAŁ AND METHODS Birds Twenty-one 2-yr-old Yangzhou goose ganders (Anser cygnoides) were kept in individual pens under natural lighting and temperature conditions, with access to a pool of water. One hundred twenty 2-yr-old female geese from a cross of 2 breeds (Lijia and Zi geese, A. cygnoides) were randomly divided into 4 groups of 30 individuals each and kept in separate open-air pens. Pools of water were also made available to the females. All birds were fed 250 to 300 g/d of a commercial ration for goose breeding containing 10.80 MJ of ME and 140 g of CP/kg.
Semen Collection and Determination of Semen Quality The ganders used in this study were selected on the basis of a positive reaction to dorsa-abdominal massage resulting in semen ejaculation. Semen was collected 3 times a week during the middle of the reproductive season (February). Within 30 min after collection, ejaculate volume, motility, concentration, and the morphology of spermatozoa were evaluated. The morphology of spermatozoa was evaluated after nigrosin-eosin staining (Bakst and Cecil, 1997). In each case, 300 spermatozoa per slide (1,600× magnification) were evaluated. Sperm concentration was measured by a hemocytometer, and motility was estimated by using the hanging drop method at 400×
magnification (Bakst and Cecil, 1997). The pH was determined with an EcoScan 5 pH meter (Eutech Instruments Pte Ltd., Singapore), and the volume of semen was measured by a semen collection cup to the minimum 10 L. Semen quality factor values were calculated according to the following equation (Łukaszewicz and Kruszynski, 2003): SQF = ejaculate semen volume (for individual gander evaluation; mL) or insemination dose (for diluted pooled semen evaluation; mL) × sperm concentration (×106/mL) × live and morphologically normal spermatozoa (%).
Semen Treatment Part of the freshly collected semen from each gander was used in determining semen quality, whereas the remainder was centrifuged at 800 × g for 10 min to collect the seminal plasma. Samples of seminal plasma were stored at −20°C until assayed.
Fertilizing Ability Test Based on the SQF of the last 3 evaluations, the 21 ganders were divided into 3 groups: high (H) SQF (n = 4, range from 117.8 to 264.3), medium (M) SQF (n = 7, range from 75.5 to 117.0), and low (L) SQF (n = 10, range from 20.2 to 68.7). The consideration of sufficient insemination volume needed for 30 geese resulted in uneven sample sizes for each group. Pooled semen of the H SQF group was diluted either 1:1 or 1:2 (semen:diluent) with Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen Corp., Carlsbad, CA). The remaining 2 SQF semen groups were diluted 1:1. In the end, there were 4 groups of diluted semen, namely, H(1:1), M(1:1), L(1:1), and H(1:2). The 4 groups of female geese were randomly inseminated with the 4 groups of diluted semen within 30 min of semen collection. Each goose was inseminated with 0.1 mL of diluted semen. The SQF for pooled semen was also calculated for each group. Two inseminations were performed within an interval of 5 d. Hatching eggs were collected daily from d 2 after the first insemination to d 6 after the last insemination. The eggs collected in 11 d were set in an automatic incubator and incubated at 37.8°C. Fertility was determined by candling on d 10 of incubation.
Hormone Measurements Seminal plasma testosterone and estradiol concentrations were determined by competitive immunoassay with an Immulite system (Diagnostic Products Corp., Los Angeles, CA). The intra- and interassay CV were 7.1 and 6.3% for estradiol, and 7.5 and 7.7% for testosterone, respectively. The sensitivities of the assays were 55 pmol/ L for estradiol and 0.5 nmol/L for testosterone. The im-
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field tests. The SQI is measured in 20 s at room temperature, but a sperm mobility phenotype is not determined by the SQI (Froman et al., 2003). Because of significant differences in the quantity and quality of ejaculates among individual ganders, the semen quality factor (SQF) has been proposed as a predictor of gander semen fertilizing ability (Łukaszewicz and Kruszynski, 2003). The SQF is a composite of 3 important semen traits: semen volume, semen concentration, and the percentage of live and morphologically normal spermatozoa. High-quality semen relies on normal spermatogenesis. It is becoming increasingly evident that estrogen, in addition to testosterone, plays a role in the development and function of the testis and male reproductive tract (Rivas et al., 2002; Akingbemi, 2005). The hormone level in seminal plasma is a direct reflection of male testicular endocrine activity. The relationships between semen quality and concentration of testosterone in avian seminal plasma have been discussed (Zeman et al., 1986; Cecil and Bakst, 1988). Although many sperm quality indices are used in evaluating avian semen quality, a method that accurately predicts fertility and is feasible to apply under field conditions is lacking. In the current study, the suitability of SQF in evaluating gander fertility was explored, and the roles of sex hormones in gander reproductive function were examined.
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RESEARCH NOTE Table 1. Descriptive statistics of semen quality parameters Item Mean ± SEM Range CV (%)
1
Volume (mL)
Concentration (×106/mL)
Live and normal morphology (%)
Motility (%)
pH
SQF2
0.41 ± 0.05 0.18–0.90 51.21
701 ± 82.3 230–2020 53.49
33.9 ± 2.3 28.0–51.7 30.38
54.2 ± 1.8 40.1–67.0 15.68
7.10 ± 0.02 7.02–7.21 1.41
84.0 ± 11.8 20.2–264.3 64.52
1
n = 21. Semen quality factor (SQF) = ejaculate volume (mL) × sperm concentration (×106/mL) × live and morphologically normal spermatozoa (%). 2
munoassays were validated for measurement of hormones in semen by addition of the following standards: 17β estradiol, 1:230 pmol/L, and testosterone, 1.0 nmol/L.
The levels of sex hormones were logarithmically transformed to obtain a normal distribution. The ratio of testosterone:estradiol was calculated and logarithmically transformed. Differences in the levels of sex hormones and the SQF among the 3 groups were analyzed by ANOVA, and the significance was determined by Duncan’s multiple-range tests (SAS Institute, 2001). The correlations between sex hormones and SQF were calculated with a Pearson correlation, and Spearman’s rank correlation was used to evaluate the relationship between SQF and fertility.
RESULTS Semen Quality and Fertility The SQF values of individual ganders varied from 20.2 to 264.3. Means for ejaculate volume, sperm concentration, live and morphologically normal spermatozoa, motility, pH, and SQF were 0.41 mL, 701 × 106/mL, 33.9%, 54.2%, 7.10, and 84.0, respectively (Table 1). Very large variations among ganders were found for all semen parameters except for pH. The CV for ejaculate volume, sperm concentration, live and morphologically normal spermatozoa, and motility were 51.21, 53.49, 30.38, and 15.68%, respectively, resulting in a CV as high as 64.52% for SQF. There were significant differences among the 3 gander groups (P < 0.05); the values were 155.2 ± 36.3, 86.7 ± 8.1, and 54.1 ± 7.3, respectively. The SQF values based on pooled semen were 19.3, 11.3, 9.5, and 13.1 for the H(1:1), M(1:1), L(1:1), and H(1:2) semen groups, respectively (Table 2). The corresponding fertility values for the different groups of geese were 83.9, 65.6, 56.8, and 74.3% for the respective semen groups, which showed the same trend as the changes in SQF. The Spearman rank correlation coefficient between SQF and fertility was 0.985 (P < 0.01).
Testosterone and Estradiol Concentrations in the 3 Gander Groups No significant difference among the different groups was observed for the levels of testosterone and estradiol
Correlation Between SQF and Sex Hormones For all 21 samples, SQF was negatively correlated with estradiol concentration (r = −0.68, P < 0.05) and positively correlated with the ratio of testosterone:estradiol (r = 0.48, P < 0.05).
DISCUSSION Turkey production in the United States relies mainly on artificial insemination, and artificial insemination is used nearly 100% in turkey breeding (Holsberger et al., 1998; Donoghue et al., 1999). Artificial insemination in goose breeding, although not used as routinely as in turkey and chicken breeding, is a very important technique that can help to eliminate ganders with poor semen quality so as to necessitate the selection of males that are
Table 2. Effects of different semen quality factor (SQF) values on geese fertility Group1
SQF2
Eggs set3 (n)
Fertile eggs (n)
Fertility (%)
H(1:1) M(1:1) L(1:1) H(1:2)
19.3 11.3 9.5 13.1
124 96 111 109
104 63 63 81
83.9 65.6 56.8 74.3
1 (1:1) H , H(1:2) = high SQF group, in which semen was diluted in 1:1 or 1:2 with Dulbecco’s modified Eagle’s medium (DMEM), respectively; M(1:1) = medium SQF group, in which semen was diluted in 1:1 with DMEM; L(1:1) = low SQF group, in which semen was diluted in 1:1 with DMEM. 2 SQF = semen quality factor for 1 dose (0.1 mL) of pooled semen in each group measured after insemination. 3 Data for eggs collected from d 2 after the first insemination to d 6 after the last insemination.
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Statistical Analysis
in seminal plasma (Figure 1). The levels of testosterone varied from 9.3 ± 1.3 nmol/L (the H SQF group) to 6.3 ± 1.9 nmol/L (the M group) and 6.4 ± 1.2 nmol/L (the L SQF group), and the corresponding values of estradiol increased from 683 ± 119 pmol/L to 740 ± 51 pmol/L and 905 ± 130 pmol/L. The ratios of testosterone to estradiol in the 3 groups were 15.5 ± 3.3, 8.6 ± 2.2, and 8.0 ± 1.4, respectively. The ratio of testosterone to estradiol in the H group was higher than that of the L group (P < 0.05). There was no significant difference between the M group and the L group or between the H group and the M group.
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producing the highest quality semen, or to overcome the difficulties of natural mating caused by the large difference in body size between males and females as well as the low libido of ganders when crossing with females from different breeds. Fertility results for artificial insemination of geese using fresh semen vary widely in the literature. Behr and Jochims (1992) recognized that satisfactory fertility can be achieved if the quantity of insemination does not decrease below 5 million per goose, but Malis (1998) obtained only 65% fertile eggs collected from weekly goose insemination with 10 million fresh spermatozoa. The SQF was used successfully as a means to evaluate semen quality in the current experiment, highlighting the differences among semen fertilizing ability. Very large variation in ejaculate volume was observed in ganders, indicating the importance of including ejaculate volume in evaluating the reproductive potential of ganders. The SQF, which takes into account the semen volume, shows an advantage over the SQI and over mobility in screening better ganders for reproduction. The current study showed a significant positive correlation between SQF and fertility, implying that the SQF could best be used as an indicator of fertility in goose artificial insemination. The evaluation of sperm morphology does not require sophisticated facilities and equipment and can be performed under field conditions. Evaluations repeated by the same person avoid operator-dependent errors (Łukaszewicz and Kruszynski, 2003). On the basis of the specific semen quality parameters in a pooled semen sample, the operator can predict the number of insemination doses needed to achieve satisfactory fertility. Several steps of spermatogenesis are under the control of estrogen via genomic and nongenomic pathways (Carreau et al., 2006). The role of estrogen in male reproduction is, in part, related to the ratio of androgens:estrogens
ACKNOWLEDGMENTS We would like to thank Kangning Zhang from the Changzhou Siji Goose Company of Jiangsu Province, China, for his assistance in the experiment. The current research was supported in part by the National Basic Research Program of China (2006CB102102).
REFERENCES Akingbemi, B. T. 2005. Estrogen regulation of testicular function. Reprod. Biol. Endocrinol. 3:1–13. Bakst, M. R., and H. C. Cecil. 1997. Techniques for semen evaluation, semen storage, and fertility determination. Poult. Sci. Assoc., Savoy, IL. Bakst, M. R., G. Wishart, and J. P. Brillard. 1994. Oviductal spermatozoa selection, transport, and storage in poultry. Poult. Sci. Rev. 5:117–143. Behr, K.-P., and U. Jochims. 1992. Artificial insemination of geese under field conditions. Page 684 in Proc. XIX World Poult. Congr., Amsterdam, the Netherlands. Vol. 1. World’s Poult. Sci. Assoc., Netherlands Branch, Wageningen. Carreau, S. C., C. Delalande, D. Silandre, S. Bourguiba, and S. Lambard. 2006. Aromatase and estrogen receptors in male reproduction. Mol. Cell. Endocrinol. 246:65–68. Cecil, H. C., and M. R. Bakst. 1988. Testosterone concentrations in blood and seminal plasma of turkeys classified as high or low semen producers. Poult. Sci. 67:1461–1464. Donoghue, A. M. 1999. Prospective approaches to avoid flock fertility problem: Predictive assessment of sperm function traits in poultry. Poult. Sci. 78:437–443. Donoghue, A. M., D. R. Holsberger, D. P. Evenson, and D. P. Froman. 1998. Semen donor selection by in vitro sperm mobility increases fertility and semen storage in the turkey hen. J. Androl. 19:295–301. Donoghue, A. M., T. S. Sonstegard, L. M. King, E. J. Smith, and D. W. Burt. 1999. Turkey sperm mobility influences paternity in the context of competitive fertilization. Biol. Reprod. 61:422–427. Froman, D. P., E. R. Bowling, and J. L. Wilson. 2003. Sperm mobility phenotype not determined by sperm quality index. Poult. Sci. 82:496–502. Froman, D. P., and A. J. Feltmann. 1998. Sperm mobility: A quantitative trait of the domestic fowl (Gallus domesticus). Biol. Reprod. 58:379–384.
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Figure 1. Comparison of the levels of sex hormones and the ratios of testosterone:estradiol in different semen quality factor (SQF) groups. Values are means. Vertical lines represent the SEM. a,bValues without a common letter are different (P < 0.05). Data for the high SQF group (n = 4) are shown by black bars, data for the medium SQF group (n = 7) are shown by diagonally striped bars, and data for the low SQF group (n = 10) are shown by open bars.
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