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Semen characteristics of ganders kept as genetic resources conservation programme

Corresponding Author

Ewa Łukaszewicz

[email protected]

orcid.org/0000-0002-9505-0056

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

Correspondence

Ewa Łukaszewicz, Wroclaw University of Environmental and Life Sciences, Institute of Animal Breeding, Chełmońskiego 38c, 51-630 Wrocław, Poland.

Email: [email protected]

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Anna Jerysz,

Anna Jerysz

orcid.org/0000-0002-9061-3729

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

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Artur Kowalczyk,

Artur Kowalczyk

orcid.org/0000-0001-7446-0147

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

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Ewa Łukaszewicz,

Corresponding Author

Ewa Łukaszewicz

[email protected]

orcid.org/0000-0002-9505-0056

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

Correspondence

Ewa Łukaszewicz, Wroclaw University of Environmental and Life Sciences, Institute of Animal Breeding, Chełmońskiego 38c, 51-630 Wrocław, Poland.

Email: [email protected]

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Anna Jerysz,

Anna Jerysz

orcid.org/0000-0002-9061-3729

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

Search for more papers by this author

Artur Kowalczyk,

Artur Kowalczyk

orcid.org/0000-0001-7446-0147

Division of Poultry Breeding, Institute of Animal Breeding, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland

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First published: 11 April 2022

https://doi.org/10.1111/rda.14123

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Abstract

The creation of genetic reserves of domesticated animal species and breeds almost become a necessity in the recent years, but there is a question what is the value of semen of males kept ex situ in vivo as gene conservation flocks. Presented studies assessed the response to semen collection by dorso-abdominal massage and the quantitative and qualitative semen characteristics of six goose breeds (Pomorska, Garbonosa, Kuban, Landes, Roman and Slovakia) covered by the genetic resources’ protection programme. Fourteen semen collection attempts were performed per male. In each breed there were ganders with low and high sensitivity to massage. The most positive reactions were stated in Pomorska ganders (67.9%) and the least in Kuban breed (52.60%). Individual male evaluation showed that only in three breeds (Pomorska, Garbonosa and Kuban) there were individuals showing 100% susceptibility to semen collection, in some breeds only one to four positive reactions (ending with ejaculation) were noted. Results obtained indicated breed and male effect on analysed semen traits, with the exception of sperm motility. The highest number of live normal sperm (44.2% on average), sperm concentration (530 × 10⁶ ml⁻¹), the highest Semen Quality Factor (92.9) and sperm motility (50.30%) were found in semen of Kuban ganders, while the lowest values of these traits (28.7%; 230 × 10⁶ ml⁻¹; 11.4 respectively) in Slovakia ganders. The lowest sperm motility (38.3%) was observed in ganders of Roman breed, but comparing to the other breeds existing differences were not significant. Significant differences in sperm morphology between individual ganders were also observed.

1 INTRODUCTION

Preservation of the biological and genetic diversity has become a global challenge. Biodiversity is concerned with the diversity of ecosystems and species, and the interactions between them. The genetic diversity refers to the genes of different individuals of a given species and population (Guemene et al., 2015). Bird populations endangered with extinction are most often protected by the in situ method, consisting in the protection of genetic resources in their natural environment (Blesbois et al., 2007). In Hungary, the introduced conservation programmes have contributed to the recognition of 22 goose genotypes, 68% of which are native Hungarian breed (Bódi et al., 2019), and in Slovakia, the endangered breeds include, among others, the local Slovak goose (Hanusová et al., 2017; Hrnčár et al., 2012; Mindek et al., 2014). The experiments carried out by our scientific team (Łukaszewicz et al., 2021) are also in line with the assumptions set out by FAO. Their aim is to maintain the genetic reserve of poultry species by preserving their original genotypes. Actually, in Poland, the programme for protection of genetic resources covers 14 goose breeds, which have the status of endangered populations and have been included in the global genetic resources subject to protection (FAO - World Watch List, 2000).

In case of domesticated animals, the ex situ in vivo methods as gene pools preservation are commonly used, which are increasingly supported by in vitro methods. For example, in France, an intensive activity in four priority areas: (1) characterization, inventory and surveillance, (2) sustainable use and valorization, (3) conservation and (4) politics, institutions were undertaken (Guemene et al., 2015). In this regard, our research team also made the first attempts to freeze the semen of 12 goose breeds in danger of extinction (non-published results).

The starting point for the ex situ method, both in vivo and in vitro, is selection of males with breed-typical characteristics and at the same time producing high-value sperm, which can then be used for artificial insemination or subjected to freezing process and deposited in semen cryobanks (Abu et al., 2013; Blesbois et al., 2005).

According to the applicable rules, any genetic selection can be carried in the conservative flocks and the applied mating system aims to maintain the unchanged frequency of genotypes and to reduce the speed of related effect. On the other hands, due to high costs of animal maintenance, the number of individuals in one breed is limited (in the case of goose, one breed is represented by 450–600 birds), which leads to inbreeding, and as the consequence, reduces the reproductive ability of animals kept ex situ in vivo. In males, the decrease in reproductive potency is manifested mainly by a decrease in semen quality. Males susceptibility to manual semen collection procedure, their sexual libido, the appearance of intromittent (phallus protruden) copulatory organ and quality of ejaculated semen are the basic criteria for selecting the best ganders for breeding purposes (Gerzilov, 2004; Gerzilov & Nickolova, 2010; Łukaszewicz et al., 2020; Nikolova & Gerzilov, 1999). A very good indicator of semen quality is the content of normal, properly shaped, undamaged sperm (Liu et al., 2008), and which shows a high degree of mobility (Hook & Fisher, 2020). The ejaculate volume and sperm concentration are also very important from artificial insemination and semen preservation viewpoint. Therefore, semen quality characterized by Semen Quality Factor (SQF) which considers three important semen features: volume, sperm concentration and percentage of live normal sperm, is an indicator that allows for a reliable assessment and comparison of the reproductive ability of individual males (Boz et al., 2021; Gumułka & Rozenboim, 2015; Łukaszewicz, 2002; Łukaszewicz & Kruszyński, 2003).

In the presented study, we analysed the quantitative and qualitative semen traits of six goose breeds included in the Polish genetic resources conservation programme: Pomorska (Po), Garbonosa (Ga), Kuban (Ku), Landes (LsD), Roman (Ro) and Slovakia (Sl). Intentionally we focused on the basic semen traits that can be easy evaluated without access to specialized research equipment, but those which allows to assess the male fertilizing potency. Our aim was to show the differences between goose males from genetic conservative flocks, and to draw attention to the necessity of periodic assessment of such flocks, in order to avoid excessive deterioration of semen quality.

2 MATERIAL AND METHODS

2.1 Birds and their maintenance

Fifteen 1-year-old ganders of six goose breeds included in the genetic resources’ conservation programme, managed in the Waterfowl Genetic Resource Station of the National Research Institute of Animal Production in Dworzyska, Poland, were selected randomly for experimental purposes. As a result of the preliminary assessment of the degree of copulatory organ development and cloaca appearance, and the manner of reaction to semen collection, the following number of males were left for further research: two Polish local breeds: Pomorska (Po - n = 12), Garbonosa (Ga - n = 15), and four foreign breeds: Kuban (Ku - n = 9), Landes (LsD - n = 13), Roman (Ro - n = 11) and Slovakia (Sl - n = 12). Selected males expressed erection as the reaction to massage procedure, had a properly developed phallus protrudens (phallus length at least 5–6 cm), and had any lesions in the cloaca indicating inflammation of the copulatory organ. During the experimental period (from January till the half of April) males were kept individually in spacious wire mesh boxes with a deep litter floor. Boxes were allocated in an unheated room, with natural temperature and light environment and gravity ventilation. Every day 350–400 g of commercial food (Tasomix, Biskupice Ołoboczne, Poland) for reproductive goose flocks was provided per one male and free access to fresh, good quality water (Łukaszewicz et al., 2021).

2.2 Semen collection and evaluation

Semen was collected into calibrated glass tubes which shape and size was adjusted to the size of ganders’ copulatory organ. From each male semen was collected individually twice a week (from two breeds in one day), with the use of dorso-abdominal massage (Chełmońska, 1967). Fourteen semen collections were performed from each male. Only clean semen free of uric or faecal contamination was subjected to laboratory analysis that was performed within 15–20 min after collection.

Ejaculate volume was assessed with an accuracy of 0.01 ml using automatic pipettes (Eppendorf Research^® plus) with disposable Eppendorf tips; sperm concentration was calculated by spectrophotometry (Accucel photometer, IMV Technologies, France) and sperm integrity and morphology (300 cells per slide = 100%) was examined in nigrosin–eosin smears and evaluated at 1250× under a light microscope (Jenaval, Carl Zeiss, Jena, Germany). Sperm were attributed to seven categories: six of them, named ‘total live’, were as follows: live morphologically normal (typical spindle-shaped head and well-marked acrosome); bulb-head; crooked-neck; midpiece deformed (swelling, ragged or lack of midpiece); spermatids (immature forms); spermatozoa with other deformities (not included in any of the previous category; dead spermatozoa – partly or full stained by eosin; all abnormal (damaged cells) were also summarized in one ‘category’.

As mentioned in the ‘Introduction’ section, for better comparison and scoring the differences in semen quality between individual ganders the semen quality factor (SQF) was calculated according to the following pattern:

SQF = [sperm concentration per ml × ejaculate volume (ml) × live normal spermatozoa (%)]/100%.

Sperm motility was assessed in percentage, from 10% to 100%, by observing a drop of semen under a light microscope and 40× objective (Jenaval, Carl Zeiss Jena, Germany) (Bielański, 1972).

2.3 Statistical analysis

All variables were examined for normality and homogeneity of variance using the Shapiro–Wilk test. The results that were not normally distributed were log-transformed prior to one-way analysis of variance (ANOVA). If the transformation did not change the distribution of variables, the Kruskal–Wallis test was used, while for normal distribution the Tukey post-hoc test for unequal counts was applied. All data were analysed with Statistica, version 12.5 (StatSoft, Inc., Kraków, Poland).

3 RESULTS

3.1 Ganders’ reactions to semen collection procedure

In total 1048 semen collection attempts were performed, ranging from 168 to 210, depending on ganders’ number in the particular breed. More positive reactions, that is, those ended with semen ejaculation, were found in both local Polish groups when compared to foreign breeds.

The highest percentage of positive reactions was noted in Pomorska ganders (67.86%), and the lowest in Kuban goose (52.60%) (Table 1). In each breed there were individuals with varying sexual libido. The number of positive reactions ranged from 1 (7.14%; 5 males: Ga15, Ku 53 and 55, LsD66 and Ro20) to 14 (100%; 9 males: Po4, 33, Ga13,18,44, Ku57, 60.64.65). In both Polish local breeds (Po and Ga) and one foreign (Sl), all males responded positively to manual semen collection (Tables 2–7).

TABLE 1. Fresh semen characteristics of six goose breeds involved in Polish genetic resources conservation programme (means; SEM)

Goose breed/number of males	Obtained semen samples [no] (%)	Ejaculate volume [ml]	Sperm concentration [n × 10⁶ml^−l]	Sperm morphology [%]									SQF†	Motility [%]
Goose breed/number of males	Obtained semen samples [no] (%)	Ejaculate volume [ml]	Sperm concentration [n × 10⁶ml^−l]	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deformat.	Dead sperm	Deform. together		SQF†	Motility [%]
Polish local breeds
Pomorska n = 12	114‡ (67.9%)	0.25^b	270^b	90.8^bc	40.9^a	23.8^c	4.5^b	12.5^a	9.1^b	9.2^bc		45.4^b	29.4^bc	44.9
Garbonosa n = 15	128 (61.0%)	0.32^a	510^a	91.2^b	32.5^c	29.8^a	7.8^ab	7.8^c	13.4^a	8.8^c		51.0^a	53.5^ab	49.5
Foreign breeds
Kuban n = 9	81 (64.3%)	0.40^a	530^a	93.3^a	44.2^a	22.2^c	11.0^a	9.4^bc	6.5^c	6.6^d		38.1^c	92.9^a	50.3
Landes n = 13	101 (55.5%)	0.25^b	220^b	90.4^bcd	38.1^ab	25.1^bc	4.3^b	13.0^a	10.0^b	9.6^abc		48.2^ab	23.5^bc	39.4
Roman n = 11	94 (61.0%)	0.18^b	260^b	88.0^d	32.9^bc	26.8^ab	7.4^ab	11.9^ab	8.7^b	12.0^a		47.4^ab	15.7^c	38.3
Slovakia n = 12	95 (56.6%)	0.17^b	230^b	88.5 cd	28.7^c	31.5^a	9.0^a	9.0^bc	9.4^b	11.5^ab		49.9^a	11.4^c	47.1
SEM	--	0.014	0.028	0.233	0.586	0.379	0.314	0.226	0.217	0.233		0.471	0.525	1.616
Genotype effect	--	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001		0.001	0.001	0.129

^a…dMeans in columns followed by different superscripts differ significantly (p < .05).
^† SQF – Semen Quality Factor.
^‡ For every male 14 semen collection attempts were performed; the number of attempts from particular breed depended on ganders’ number in the given breed.

TABLE 2. Characteristics of the fresh semen collected individually from Pomorska (Po) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
Po 1	12† (85.71)	94.7^a	54.1^a	19.8^b	9.2^a	5.6^b	6.1^b	5.2^b	31.4^c
Po 4	14 (100)	89.5^ab	43.0^ab	23.9^b	1.5^b	13.3^a	7.9^ab	10.5^ab	45.0^abc
Po 6	^x5 (35.71)	96.0	46.4	28.7	3.8	6.9	10.3	4.0	45.8
Po 22	13 (92.86)	87.2^b	41.7^ab	22.0^b	2.2^b	15.4^a	5.9^b	12.8^a	43.3^abc
Po 25	^x3 (21.43)	93.7	43.1	18.1	11.3	8.7	12.4	6.3	39.2
Po 29	10 (71.43)	87.8^b	45.5^ab	24.3^b	1.7^b	8.0^b	8.3^ab	12.2^a	40.6^bc
Po 30	^x4 (28.57)	91.2	45.2	23.2	2.7	6.7	13.4	8.8	43.2
Po 31	10 (71.43)	90.5^ab	20.1^c	31.9^a	11.5^a	15.8^a	11.3^a	9.5^ab	59.0^a
Po 33	14 (100)	90.6^ab	41.5^ab	19.6^b	2.1^b	15.6^a	11.8^a	9.4^ab	47.0^ab
Po 35	13 (92.86)	89.5^ab	38.7^abc	21.5^b	1.7^b	16.6^a	11.0^a	10.5^ab	49.0^ab
Po 37	^x5 (35.71)	94.2	52.9	21.1	3.3	11.7	5.3	5.8	38.1
Po 38	11 (78.57)	92.3^ab	29.3^b	31.8^a	7.8^a	14.1^a	9.4^ab	7.7^ab	55.2^ab
SEM	--	0.507	1.303	0.853	0.383	0.477	0.390	0.507	1.19
Male effect	--	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001

^a…cMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

TABLE 3. Characteristics of the fresh semen collected individually from Garbonosa (Ga) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
Ga 12	11† (78.57)	90.8^abc	31.7^cd	38.6^ab	4.6^cd	6.7^b	9.1^cd	9.2^abc	54.4^ab
Ga 13	14 (100.0)	95.2^a	40.8^bc	32.7^b	5.2^bcd	5.4^b	11.1^bcd	4.8^c	49.2^b
Ga 14	^x4 (28.57)	91.5	47.2	22.1	4.0	7.7	10.4	8.5	40.2
Ga 15	^x1 (7.14)	93.0	29.0	33.0	1.0	7.7	22.3	7.0	63.0
Ga 16	13 (92.86)	88.5^c	25.8^de	22.5^c	15.7^a	6.4^b	18.0^ab	11.5^a	46.9^bc
Ga 17	13 (92.86)	89.7^bc	13.9^e	29.3^bc	12.8^ab	14.2^a	19.5^ab	10.3^ab	63.1^a
Ga 18	14 (100.0)	94.5^ab	54.5^a	23.4^c	2.3^d	8.2^ab	6.1^d	5.5^bc	37.7^c
Ga 41	^x2 (14.29)	90.3	43.2	19.8	10.3	6.5	10.5	9.7	36.8
Ga 42	^x4 (28.57)	90.4	12.7	28.6	21.7	6.2	21.2	9.6	56.1
Ga 43	^x3 (21.43)	91.7	28.1	34.1	6.2	8.6	14.7	8.3	57.3
Ga 44	14 (100.0)	89.5^bc	28.1^d	29.6^bc	12.8^ab	6.1^b	12.9^abc	10.5^ab	48.7^b
Ga 49	9 (64.29)	89.4	24.8^de	20.4^c	9.4^abc	7.2^ab	27.6^a	10.6^ab	55.1^ab
Ga 51	6 (42.86)	90.4^abc	28.3^cd	30.8^b	4.8^bcd	15.6^a	11.0^bcd	9.6^abc	57.4^ab
Ga 54	12 (85.71)	87.5^c	24.9^de	42.6^a	2.5^d	6.6^b	10.8^bcd	12.5^a	60.1^a
Ga 59	9 (64.29)	96.4^a	51.2^ab	31.8^b	1.9^d	5.4^b	6.1^d	3.6^c	43.3^bc
SEM	--	0.473	1.461	0.898	0.522	0.377	0.702	0.473	1.059
Male effect	--	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001

^a…eMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

TABLE 4. Characteristics of the fresh semen collected individually from Kuban (Ku) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
Ku 53	^x1† (7.14)	84.3	23.7	23.0	2.7	16.3	18.7	15.7	58.0
Ku 55	^x1 (7.14)	95.3	34.3	13.0	37.0	5.7	5.3	4.7	24.0
Ku 57	14 (100.0)	93.9	46.5^bc	24.1^ab	3.0^c	14.0^a	6.3	6.1	44.4^a
Ku 58	6 (42.86)	90.6	35.0^cd	18.1^b	16.4^b	11.6^abc	9.7	9.4	39.2^ab
Ku 60	14 (100.0)	94.6	23.4^d	18.6^b	41.3^a	5.5^e	5.8	5.4	29.9^b
Ku 61	6 (42.86)	90.5	46.5^abc	20.9^ab	2.3^c	13.5^ab	7.3	9.5	41.7^ab
Ku 63	11 (78.57)	95.0	61.9^a	20.3^ab	1.6^c	5.8^de	5.3	5.0	31.5^b
Ku 64	14 (100.0)	92.9	50.4^ab	23.4^ab	2.4^c	9.4^bcd	7.3	7.0	40.1^ab
Ku 65	14 (100.0)	93.5	47.9^abc	27.4^a	4.9^c	8.4^cde	5.1	6.5	40.7^ab
SEM	--	0.430	1.547	0.793	1.689	0.462	0.326	0.430	0.963
Male effect	--	0.117	0.001	0.007	0.001	0.001	0.019	0.117	0.001

^a…eMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

TABLE 5. Characteristics of the fresh semen collected individually from Landes (LsD) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
LsD 45	13† (92.86)	91.3^ab	36.8^bc	28.1^a	8.3^a	8.4^c	9.7^bc	8.7^bc	46.2^ab
LsD 46	10 (71.43)	91.5^ab	51.9^ab	18.4^b	2.7^cd	10.4^bc	8.1^bcd	8.5^bc	36.9^bc
LsD 66	^x1 (7.14)	87.3	51.3	19.3	2.7	8.7	5.3	12.7	33.3
LsD 67	12 (85.71)	93.0^ab	42.3^ab	22.2^ab	2.0^d	19.3^ab	7.1^cd	7.0^bc	48.7^ab
LsD 68	^x5 (35.71)	90.1	42.5	18.5	3.0	14.3	11.9	9.9	44.7
LsD 69	9 (64.29)	84.3^c	23.6^c	27.9^ab	5.5^b	11.0^bc	16.3^a	15.7^a	55.2^a
LsD 70	13 (92.86)	90.2^abc	24.5^c	31.3^a	5.0^bc	20.2^a	9.2^bc	9.8^abc	60.7^a
LsD 71	^x5 (35.71)	91.3	31.7	28.9	1.8	12.9	15.9	8.7	57.8
LsD 72	13 (92.86)	94.4^a	55.9^a	23.3^ab	4.3^bc	5.8^c	5.1^d	5.6^c	34.2^c
LsD 73	^x2 (14.29)	88.7	35.0	22.7	0.3	14.3	16.3	11.3	53.3
LsD 74	11 (78.57)	88.2^bc	35.5^bc	25.7^ab	5.3^b	9.8^c	11.9^ab	11.8^ab	47.4^ab
LsD 75	^x4 (28.57)	86.8	20.6	19.7	2.7	29.7	14.1	13.2	63.5
LsD 76	^x3 (21.43)	89.4	39.3	27.4	4.8	11.2	6.7	10.6	45.3
SEM	--	0.494	1.458	0.833	0.280	0.650	0.509	0.494	1.237
Male effect	--	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001

^a…dMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

TABLE 6. Characteristics of the fresh semen collected individually from Roman (Ro) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
Ro 2	9† (64.29)	87.9^ab	41.1^ab	24.0	4.6^c	10.1^cd	8.0^ab	12.1^ab	42.1^b
Ro 3	^x5 (35.71)	76.8	20.7	28.6	11.5	6.3	9.7	23.1	44.6
Ro 5	10 (71.43)	87.7^ab	21.9^c	28.7	16.0^a	10.6^c	10.6^ab	12.2^ab	49.9^ab
Ro 9	11 (78.57)	92.4^a	37.6^abc	31.2	2.9^c	14.2^bc	6.4^b	7.6^b	51.9^ab
Ro 11	11 (78.57)	91.6^a	30.1^abc	31.0	12.0^b	5.2^e	13.3^a	8.4^b	49.4^ab
Ro 19	^x4 (28.57)	79.6	29.7	23.5	1.2	11.2	14.0	20.4	48.7
Ro 20	^x1 (7.14)	83.7	37.7	20.3	6.0	14.0	5.7	16.3	40.0
Ro 32	12 (85.71)	87.3^ab	39.1^ab	24.5	4.0^c	11.3^c	8.5^ab	12.7^ab	44.2^b
Ro 39	8 (57.14)	82.2^b	25.4^bc	19.1	13.3^ab	17.1^b	7.3	17.8^a	43.5^b
Ro 47	9 (64.29)	92.2^a	27.2^bc	29.0	3.0^c	27.2^a	5.8^b	7.8^b	62.0^a
Ro 50	12 (85.71)	89.8^ab	45.2^a	26.8	4.1^c	6.7^de	7.0^b	10.2^ab	40.5^b
SEM	--	0.702	1.409	1.024	0.598	0.720	0.416	0.702	1.205
Male effect	--	0.001	0.001	0.102	0.001	0.001	0.001	0.001	0.001

^a…eMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

TABLE 7. Characteristics of the fresh semen collected individually from Slovakia (Sl) goose covered by the genetic resources protection programme (means; SEM)

Gander's number	Obtained samples [no] (%)	Sperm morphology [%]
Gander's number	Obtained samples [no] (%)	Total live	Live normal	Bulb head	Spermatids	Bent neck	Other deform.	Dead sperm	Deform. together
Sl 10	7† (50.00)	97.3^a	27.8^abc	26.2^ab	31.6^a	6.1^bc	5.6^b	2.7^b	38.0^b
Sl 21	10 (71.43)	84.3^b	27.4^bc	28.4^ab	11.7^b	5.0^c	11.8^a	15.7^a	45.2^ab
Sl 23	^x5 (35.71)	92.1	26.5	37.2	10.3	6.9	11.2	7.9	55.3
Sl 24	^x5 (35.71)	88.4	24.0	37.4	7.6	8.7	10.7	11.6	56.8
Sl 26	10 (71.43)	85.3^b	41.6^a	22.3^b	3.2^e	9.6^ab	8.6^ab	14.7^a	40.5^b
Sl 27	11 (78.57)	83.9^b	18.7^c	36.2^a	7.1^cd	11.1^a	10.8^ab	16.1^a	58.1^a
Sl 28	6 (42.86)	92.6^ab	35.9^ab	29.7^ab	4.6^de	9.8^ab	12.6^a	7.4^ab	52.1^ab
Sl 34	7 (50.00)	95.7^a	24.9^bc	36.1^a	13.3^b	12.6^a	8.9^ab	4.3^b	57.6^a
Sl 36	^x4 (28.57)	87.9	28.6	29.2	4.8	13.5	11.8	12.1	54.5
Sl 40	12 (85.71)	84.1^b	24.7^bc	29.3^ab	10.2^bc	11.7^a	8.1^ab	15.9^a	49.1^ab
Sl 48	10 (71.43)	90.5^ab	34.7^ab	36.6^a	5.7^de	5.5^bc	8.0^ab	9.5	50.0^ab
Sl 52	8 (57.14)	89.0^ab	30.0^ab	33.4^ab	9.4^bcd	9.0^abc	7.2^ab	11.0^ab	49.6^ab
SEM	--	0.761	1.155	1.010	0.753	0.416	0.388	0.761	1.101
Male effect	--	0.001	0.001	0.009	0.001	0.001	0.002	0.001	0.001

^a…eMeans in columns followed by different superscripts differ significantly (p < .05).
^xFive or less histological smears for sperm morphology evaluations were obtained therefore the results were not considered in the statistical analysis.
^† For every male 14 semen collection attempts were performed.

3.2 Semen evaluation

The statistical analysis of semen characteristics of all analysed goose breeds is presented in Table 1, while of individual males within particular breed in Tables 2–7.

The effect of male genotype on semen volume, sperm concentration and morphology, as well as on SQF was observed. The average semen volume varied from 0.25 ml (Po breed; 0.19–0.31 ml) to 0.32 ml (Ga; 0.27–0.38 ml). In foreign breeds it ranged from 0.17 ml (Sl, 0.17–0.18 ml), 0.18 ml (Ro, 0.10–0.21 ml), 0.25 ml (LsD, 0.20–0.32 ml) to 0.40 ml (Ku, 0.25–0.53 ml). The volume of Kuban gander semen was similar to that of Ga (p ≥ .05), and compared to the other groups, it was significantly higher (p ≤ .05).

The average sperm concentration in the Polish breeds ranged from 270 × 10⁶ml⁻¹ (Po; from 210 to 380 × 10⁶ ml⁻¹) to 510 × 10⁶ ml⁻¹ (Ga; 250–1070 × 10⁶ml⁻¹), while in foreign goose breeds from 220 × 10⁶ml⁻¹ (LsD; 60–320 × 10⁶ml⁻¹), 230 × 10⁶ml⁻¹ (Sl; 10–390 × 10⁶ ml⁻¹), 260 × 10⁶ ml⁻¹ (Ro; 70–380 × 10⁶ ml⁻¹) to 530 × 10⁶ ml⁻¹ (Ku; 310–850 × 10⁶ ml⁻¹). In case of this sperm characteristic, statistically significant differences (p ≤ .05) were stated, only between the Ku and Ga ganders, compared to the other breeds.

The highest value of SQF (92.9) was observed for Kuban gander (extreme values: 57.3–164.3), which differed significantly (p ≤ .05) from all remained groups, while the lowest (11.4; 4.8–20.6) for Slovakia ganders. Among two Polish local breeds Ga ganders had higher SQF value (p ≤ .05) than Po ganders (53.5 on average; 30.1–109.4 and 29.4; 25.5–45.8 respectively).

There was no genotype effect on sperm motility. The best motility (50.3% on average; 30.0%–65.6%) was stated in Kuban gander semen, and the lowest in Roman ganders (38.3%; 10.0%–50.0%). In ganders of the remaining breeds sperm motility was within 25.0%–61.1%.

Goose breed (Table 1) and ganders of particular breed (Tables 2–7) effect on sperm morphological structure was observed, except live in total and dead sperm content in Kuban gander semen (Table 4) and bulb head sperm in Roman breed (Table 6).

The highest, average content of total live sperm (93.3%; ranging from 81.7% to 99.3%) was stated in Ga gander semen, which differed significantly (p ≤ .05) from the results of all remained breeds. In semen of Po, Ga and LsD ganders the percentage of total live sperm was on a similar level, oscillating around 91% (73.0%–99.0%), while in Ro and Sl breeds about 88.0% (from 69.7% to 98.7% and from 44.3% to 99.7% respectively).

The highest number of live normal sperm (44.2% on average; from 8.3% to 69.0%; Table 4) contained Kuban gander semen and their content was significantly (p ≤ .05) higher compared to other breeds (Table 1). The smallest number of this form of sperm (from 4.0% to 55.3%) was observed in Slovakia males (Table 7). Among the assessed males, the Kuban male no. 63 had the most valuable semen which had, on average 61.9% of normal sperm (from 36.0% to 69.0%; Table 4), and the poorest (only 12.7 of normal cells, ranging from 6.0% to 20.0%) Ga42 (Table 3).

Among the analysed goose breeds, the highest amount of deformed sperm together (51.0%; Table 1) was observed in Ga gander semen, but this value did not differ significantly (p ≥ .05) from Sl and LsD gander semen. The least deformed sperm (p ≤ .05) contained the sperm of Kuban ganders (Table 1). For the individual males, the percentage of deformed sperm varied from 29.9 (♂Ku60; 22.0–42.0; Table 4) to 63.1 (♂Ga17; 58.0–72.0; Table 3).

Bulb head sperm predominated in the population of abnormal (deformed) forms, followed by bent neck (Table 1). The highest amount of bulb head sperm was observed in Sl (31.5% on average, Table 1; ranging from 10.7% to 57.0%, Table 7) and Ga gander semen (29.8%, Table 1; 12.7%–62.7%, Table 3). For both breeds these values were significantly (p ≤ .05) higher compared to Kuban ganders (22.2%, Table 1; 7.7%–42.0%; Table 4). The mean content of distended sperm in the male ejaculates ranged from over 18% (Po25, Ku58 Ku56, LsD46, LsD68) to 42.6% (♂Ga54) (Tables 2–7).

The highest number of sperm with a bent neck (at the head–midpiece junction, the neck was bent at acute angle), was in LsD ganders (3.3%–36.0%; Table 5), and their average content (13.0%; Table 1) was insignificantly (p ≤ .05) higher than in Po and Ro ganders. The smallest number of bent neck sperm was stated in Ga gander semen (7.8% and 1.7%–24.7% respectively) (Table 1 and Table 3). In semen collected from individual males, the average content of bent neck sperm ranged from 5.0%–5.8% (Po1, Ga13, Ga59, Ku60, Ku63, LsD72, Ro11 and Sl21.48) to 20.2% (LsD70) (Tables 2–7).

It should be noted that apart from live normal and damaged forms, the spermatids – immature forms (most often they were very mobile and viable, round cells, with a thin flagellum, occasionally a slightly elongated spermatids with a cytoplasmic drop on the flagellum were observed, indicating an unfinished transformation process into a sperm), made up a significant proportion in semen of assessed goose breeds. The highest number of spermatids was found in semen of Kuban (11.0% on average; 0.0%–50.0%) and Slovakia (9.8%; 0.0%–36.3%) ganders, which differed significantly (p ≤ .05) from their amount in Po (4.5%; 0.0%–17.3%) and LsD (4.3%; 0.0%–14.0%) ganders.

4 DISCUSSION

In the discussed studies, we confirmed a diversified susceptibility of ganders to semen collection procedure, as well as the breed and individual male effect on semen quality. Some males responded to manual massage with 100% efficiency, and some of them either did not respond to massage at all (0%) or at a very low level (7.1%–28.6%). This confirms the opinion of other authors (Liu et. al., 2008; Łukaszewicz et. al., 2000; Nikolova & Gerzilov, 1999), that males intended as semen donors should be previously assessed for the response to semen collection procedure, and only these individuals from which good quality ejaculates can be obtain regularly in a subsequent collection should be left.

In this study we observed more positive responses to massage in all breeds compared to the results obtained in our previous studies on Polish regional goose Kartuska and Rypińska (36.4% and 44.3%), while the reactions of foreign breeds were similar to Suwalska, Kielecka, Lubelska and Podkarpacka geese (57%–58.6%) (Łukaszewicz et al., 2021). Nevertheless, in all analysed breeds, the ganders’ susceptibility to massage was much lower than of commercial line White Koluda^® (nearly 83%; Łukaszewicz & Kruszyński, 2003) or Canada goose (Branta canadensis; 61%; Zaik, 2013). It is difficult to define unequivocally what influences ganders’ reaction to semen collection procedure, certainly individual properties. In case of the commercial line as White Koluda^®, maybe long lasting selection toward improvement of the reproductive traits? Goose breeds derived from Anser cygnoides generally have higher reproductive indices, so it is possible that they also respond better and faster to massage, comparing to Anser anser derived breeds. Zaik (2013) described that in the analysed male population there were individuals that always responded to massage, but did not always ejaculate semen. Such reactions were not observed in our studies. Nikolova and Gerzilov (1999) showed that, due to poor or no reaction, they had to discard from further studies 50% of ganders intended for reproduction. A significantly lower percentage of positively reactions of Zi (Anser cygnoides L.) and Rhin (Anser anser L.) ganders (30% and 46% respectively) were described by Liu et al. (2014), and Hungarian ganders (30%) – Almasi et al. (2002) and Varga et al. (2003). A varied level of reaction has also been observed in males of other bird species (Gerzilov, 2004; Kowalczyk & Łukaszewicz, 2012; Kowalczyk et al., 2012; Liu et al., 2014; Łukaszewicz et al., 2020).

In the presented experiment, as in studies of other authors, a large breed and individual male variations in quantitative and qualitative semen traits were observed. The obtained average semen volumes (0.18–0.40 ml) are comparable with other regional goose breeds (0.16–0.41 ml, Łukaszewicz et al., 2021), but definitely lower than those of White Roman gander in Chang et al. (2016) study. Diverse ejaculate volumes were described by other authors for the same or different goose genotype. For example, the average volume of White Koluda^® semen ranged from 0.20 ml (Jerysz & Łukaszewicz, 2013) to 0.32 ml (Łukaszewicz & Kruszyński, 2003), Canadian goose – from 0.21 to 0.25 ml (Kowalczyk & Łukaszewicz, 2012), Zi and Rhin geese – 0.42 and 0.31 ml respectively (Liu et al., 2014), Yangzhou goose – 0.41 ml (Liu et al., 2008), native Turkish goose – 0.24 ml (Boz et al., 2021 ) and finally Egyptian goose – from 0.61 to 0.51 ml, depending on housing system (El-Hanoun et al., 2012).

The genotype of ganders influenced sperm concentration, which varied from 220 × 10⁶mL⁻¹ in Landes to 530 × 10⁶mL⁻¹ in Kuban ganders (Table 1). In the earlier studies carried out on goose covered by the genetic resources’ conservation programme the average sperm concentration in Kuban gander was 670 000 in 1 mm³ (Chełmońska et al., 1984), Zatorska ganders from 137.6 × 10⁶mL⁻¹ to 331.2 × 10⁶mL⁻¹ (Gumułka & Rozenboim, 2015), in semen of Biłgorajska goose 190.0 × 10⁶mL⁻¹ (Opałka et al., 2008) and in White Roman from 0.6 to 1.3 × 10⁸ml⁻¹ (Chang et al., 2016). In studies conducted on the other goose species Łukaszewicz et al. (2004) found 35.36 × 10⁶mL⁻¹ sperm on average in Greylag (Anser anser L.) gander semen, Kowalczyk and Łukaszewicz (2012) – 213.4 × 10⁶mL⁻¹ and Zaik (2013) – 231 × 10⁶mL⁻¹ in wild Canada goose semen, Boz et al. (2021) – 341 × 10⁶mL⁻¹ in native Turkish goose, and El-Hanoun et al. (2012) in the Egyptian goose from 686 to 798 × 10⁶mL⁻¹.

In the presented study, sperm motility was not dependent on ganders’ origin; however, the highest number of sperm showing progressive movement (50.3%) was noted in Kuban gander semen and the lowest (38.26%) in Roman breed. Compared to our results Dubos et al. (2006) described more progressive moving sperm in Landes gander semen (3–3.5 points on a 6-point scale, what means over 50% motility) and Nikolova and Gerzilov (1999; 79.39% and 67.78%). Similar or lower values in White Roman goose semen described Chang et al. (2016), who reported also that depending on light type in goose building, sperm motility amounts 25.6%–44.0%. Sperm motility in goose breeds analysed in this experiment was better than in Rhin goose semen (37.4%) (Liu et al., 2014). Almasi et al. (2002) and Varga et al. (2003) reported that sperm motility in frizzled Hungarian gander semen is over 2 points (on a 3-point scale), which corresponds to 35%–70% of sperm showing movement. In our study, we did not find the male genotype effect on sperm motility, and the differences shown by other authors may result from species, time that has elapsed since semen collection, semen storage temperature and the adopted evaluation criterion.

Both, the ejaculate volume and sperm concentration depend not only on male genotype but also on environmental temperature, frequency and skill in semen collection, and cannot be the only criterion for male evaluation. Very important, from fertilizing ability viewpoint, is sperm morphology, especially the number of live properly formed sperm, potentially having the highest fertilizing capacity. Therefore, in our experiments referred to male reproductive potency we pay particular attention to sperm structure. The integrity of cell membrane informs about sperm reaction to various unfavourable environmental conditions (Blesbois et al., 2005), and can also indicate semen quality depending on male origin. It should be emphasized that in goose, which semen contains a large number of live sperm in total, but very few sperm with normal structure and a high percentage of deformed forms, the assessment of sperm morphology is of particular importance. Determining the total number of live sperm is definitely insufficient to assess the reproductive suitability of ganders.

The significant effect of gander breed (Table 1) on sperm morphological structure has been observed, as well. These observations are consistent with those obtained by us when evaluating the semen of other regional gander breeds (Łukaszewicz et al., 2021). The most valuable was Kuban gander semen, with the highest percentage of live normal sperm (44.2%) and the lowest content of dead sperm (6.6%). The smallest number of live normal sperm (28.7%) contained Slovakia gander semen and it was significantly lower than that of White Roman (32.0%–55.1%) in Chang et al. (2016) experiment or Landes goose – 74% (Váradi et al., 2019) and 39.1% stated by Dubos et al. (2006). In studies carried out on other goose breeds endangered with extinction Gumułka and Rozenboim (2015), showed 41.9%–50.0% normal sperm in Zatorska goose, and Opałka et al. (2008) observed 69.17% of this form in Biłgorajska breed. In our previous studies, we noted 42.9% (Jerysz & Łukaszewicz, 2013) and 40.60% (Łukaszewicz, 2006) of normal sperm in White Koluda^® ganders, similar to Liu et al. (2014) in semen of Zi goose (40.8%). Kowalczyk and Łukaszewicz (2012) in Canada goose, similar as Al-Daraji et al. (2011) in local Iraqi ganders and El-Hanoun et al. (2012) in Egyptian goose described respectively, 46.3%, 51.9% and 57.1%–64.2% of normal sperm. The differences in the values obtained by cited authors may arise from many factors, one of them is bird origin. Geese bred today are derived mainly from two ancestors: Swan goose (Anser cygnoides) and Greylag (Anser anser), and commercial breeds and lines derived from these ancestors differ in performance and reproductive traits (Łukaszewicz, 2010). Individual evaluation of ejaculates showed that the semen with more than 50% of live normal sperm was produced only by 12.5% of assessed ganders (9 individuals out of 72 evaluated) and only in four breeds (Po, Ga, Ku and LsD). Among the deformed sperm, the highest percentage constituted bulb head sperm (from 7.0% to 53.0% in the individual ejaculates), then with bent neck (7.0%–62.7%). The available literature data and own research show that bent neck (crocked-neck) sperm seems to be the most common defect in bird semen. Contrary to our observations, Almasi et al. (2002) and Varga et al. (2003) showed that in ejaculates of individually assessed frizzled Hungarian ganders, sperm with ‘acrosome anomaly’ were the most numerous among deformed sperm. In Kuban breed, which semen had the highest percentage of normal sperm (44.2%) and the highest SQF index (92.9), we observed also the highest number of immature cells (11.0%, on average). This result was probably influenced by the Ku60 male, whose semen contained from 28.7% to 50.0% of spermatids. According to Chełmońska (1972a,1972b), among all poultry species, gander semen has the lowest number of live normal sperm (15%–40%, depending on the reproductive period) and a high proportion of immature forms. This may be due to the different functioning of gander testes, compared to other poultry species (Johnson, 1954). In relation to body weight, the weight of left gander's testicle was 0.1% and the right one – 0.05%, while for the duck, it was 1.99 and 1.94 respectively (Chelmońska, 1972b). Also the histological analysis of gander testes indicated a weak spermatogenesis, manifested by a lower number of layers of the reproductive cells generation than in roosters, toms and drakes (Chełmońska, 1972b). Probably the observed, very high content of spermatids was only an individual disorder of spermatogenesis in Ku60 male.

Presented results show that even uniform flock (all evaluated males were at the same age and derived from one parental flock), may have an individual's showing either disorders in spermatogenesis process, or maturing later then other males. They also confirm the necessity to perform an initial assessment of males in terms of their suitability as semen donors.

Liu et al. (2008) stated in goose that SQF is positively correlated with fertility rate and can be used as a good indicator in goose artificial insemination. We also observed the gander origin effect on SQF, which ranged in single ejaculates from 5.6 (in Roman breed) to 164.3 in Kuban breed. In the semen of Zatorska goose, which is also included in the genetic resources’ conservation programme, Gumułka and Rozenboim (2015) noted the SQF value within the range of 11.0–25.1, while Chang et al. (2016) showed from 75.4 to 99.3 in White Roman. Due to low values of ejaculate volume, sperm concentration and percentage of normal sperm in goose semen, compared to other poultry species, the SQF values are also low. For commercial White Koluda^®, it amounted 32.71 (Łukaszewicz, 2006) and 39.6 (Łukaszewicz & Kruszyński, 2003) on average, and 20.3 for Canada goose (Kowalczyk & Łukaszewicz, 2012). Slightly higher values were shown by Liu et al. (2008) for Yangzhou – 84.0 or Zi goose – 30.4, and Rhin goose – 51.3 (Liu et al., 2014) and Boz et al. (2021) for native Turkish ganders (29.12).

The presented and discussed data showed a very large breed and individual gander diversity both, in terms of male response to manual semen collection and the quality of the obtained ejaculates. The low semen quality of goose breed kept as genetic reserve flocks indicates the necessity of periodic evaluation their reproductive traits and male selection on the basis of simple analyses used in this experiment.

ACKNOWLEDGEMENTS

This study was financially supported by the Ministry of Science and Higher Education in Poland (statutory activity) and by Grant Nr N R12 0041 06/2009. The authors would like to express warm thanks to Dr Katarzyna Czyż from Animal Breeding Institute for her kindness and the final linguistic correction of the manuscript.

CONFLICT OF INTEREST

All authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript or in the decision to publish the results.

ETHICS APPROVAL

The study was carried out in a strict accordance with the recommendations of the National Ethics Commission (Warsaw, Poland), and according to Polish law and the EU directive (no 2010/63/EU), the experiment conducted within this study did not require the formal approval of Ethics Commission. Semen collection is a routine procedure for poultry reproductive flock. All members of the research staff were trained in animal care and handling by PolLASA (Polish Laboratory Animal Science Association).

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Volume57, Issue8

August 2022

Pages 815-828

Semen characteristics of ganders kept as genetic resources conservation programme

Abstract

1 INTRODUCTION

2 MATERIAL AND METHODS

2.1 Birds and their maintenance

2.2 Semen collection and evaluation

2.3 Statistical analysis

3 RESULTS

3.1 Ganders’ reactions to semen collection procedure

3.2 Semen evaluation

4 DISCUSSION

ACKNOWLEDGEMENTS

CONFLICT OF INTEREST

ETHICS APPROVAL

REFERENCES

References

Information

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Semen characteristics of ganders kept as genetic resources conservation programme

Abstract

1 INTRODUCTION

2 MATERIAL AND METHODS

2.1 Birds and their maintenance

2.2 Semen collection and evaluation

2.3 Statistical analysis

3 RESULTS

3.1 Ganders’ reactions to semen collection procedure

3.2 Semen evaluation

4 DISCUSSION

ACKNOWLEDGEMENTS

CONFLICT OF INTEREST

ETHICS APPROVAL

REFERENCES

References

Related

Information