Effects of Various Semen Extenders on the Hatching Characteristics of Japanese Quail

ABSTRACT

Reproductive efficiency is a cornerstone of success in poultry production, particularly in species such as the Japanese quail (Coturnix coturnix japonica), which are valued for their high productivity and adaptability. Semen extenders have been evaluated for their efficacy in preserving sperm quality and improving reproductive outcomes. This study assessed the effects of different semen extenders on the reproductive performance of Japanese quail, focusing on key parameters such as fertility, hatchability, and semen quality. Over a period of twenty-eight days, a total of 300 adult female breeder quails (5 to 6 weeks of age) with an average weight of 200 ± 8 g were divided into four experimental groups, under a completely randomized design. The treatment groups included T₁ (proctodeal gland foam), T₂ (quail egg albumen), T₃ (Shit’s extenders), and T₄ (control group with undiluted semen). Each group comprised five replicates with 15 birds per replicate. Additionally, 75 male breeder quails with an average weight of 170 ± 7 g were housed separately for semen collection and analysis. The results showed that birds in the T₁ group, treated with proctodeal gland foam as a semen extender, exhibited the highest fertility and hatchability rates. This group also displayed improved semen quality, with increased sperm motility and a higher percentage of live sperm count. These findings suggest that proctodeal gland foam is an effective semen extender for enhancing reproductive outcomes in Japanese quails.

Keywords:
Fertility; hatchability; quail; semen extenders; semen evaluation

INTRODUCTION

Japanese quails, locally known as “Batair”, have recently gained prominence in Pakistan’s poultry industry (Ghayas et al., 2017; Hussain et al., 2019). The high profitability of quail farming has made it an attractive option in this sector (Arshad et al., 2021). Approximately 40 years ago, hybrid Japanese quails, recognized for their exceptional egg production and quality, were introduced to Pakistan (Jatoi et al., 2013). Despite their potential, the commercial expansion of quail farming has faced challenges, particularly due to issues related to low fertility and hatchability rates (Reddish et al., 1996; Khalifa et al., 2023). Enhancing reproductive performance in these birds is therefore a key focus for both researchers and producers, as it directly impacts the profitability and sustainability of poultry operations. Artificial insemination (AI) is a transformative tool in poultry breeding programs, optimizing reproductive performance and enhancing genetic diversity (Wang et al., 2024). By enabling the controlled use of superior genetic material, AI reduces reliance on natural mating, which can be inconsistent and less efficient. Semen extenders play a crucial role in this process, preserving sperm viability, motility, and functionality during storage and transportation (Bustani & Baiee, 2021). Typically composed of nutrients, buffers, and antimicrobial agents, these extenders create an optimal environment for sperm cells, thereby increasing the success rates of artificial insemination (Bustani & Baiee, 2021; Tvrdá et al., 2023).

Among the various options available, natural and synthetic extenders have shown varying levels of effectiveness in preserving sperm quality. For instance, egg yolk-based extenders have traditionally been utilized for their ability to protect sperm membranes from damage (Alkali et al., 2022). More recently, novel options such as proctodeal gland foam and protein-rich substrates have gained attention for their potential to enhance reproductive outcomes in quails. The proctodeal gland foam, a natural secretion from male quails, contains a unique composition of proteins, enzymes, and antimicrobial agents that may enhance sperm preservation and functionality (Farooq et al., 2015). This foam plays a critical role during copulation, facilitating sperm transfer and increasing fertilization success. As an artificial semen extender, it has shown promising results in early studies, particularly in maintaining sperm motility and viability (Farooq et al., 2015; Abuoghaba et al., 2024). Similarly, quail egg albumen has been investigated for its protective and nutritional properties, which sustain sperm cells during storage and artificial insemination (Mohan et al., 2023).

In contrast, synthetic extenders such as Shit’s extenders have been developed to provide precise control over the composition of the medium, ensuring consistent protection and optimized conditions for sperm preservation. Although synthetic extenders are widely used in livestock breeding, their efficacy in quail reproduction remains underexplored. Comparing synthetic extenders with natural alternatives such as proctodeal gland foam and egg albumen can provide valuable insights into the most effective strategies for enhancing fertility and hatchability in Japanese quails. Therefore, this study was designed to evaluate the impact of different semen extenders on the reproductive performance of Japanese quail (Coturnix coturnix japonica), with a focus on fertility, hatchability, and semen quality.

MATERIALS AND METHODS

Animal care

The experiment followed protocols approved by the Ethical Review Committee for Biomedical Research at the University of Agriculture Peshawar (UAP), Pakistan, with authorization granted by Letter No. 687-A/UAP, dated 06/01/2023.

Location and duration of the experiment

The trial took place from March to April 2023 at the Poultry Unit of the Department of Poultry Science at the University of Agriculture, Peshawar, Pakistan. The experimental site is situated at coordinates 34°N, 71°E, at an altitude of 450 m above sea level. Peshawar, the city where the trial was conducted, has a typically hot and humid tropical climate, with temperatures ranging from 5°C in the winter to +45°C in the summer. The study lasted for four weeks.

Experimental birds and husbandry

A total of 300 adult female breeder quails (5 to 6 weeks of age) with an average weight of 200 ± 8 g were divided into four experimental groups under a completely randomized design. The treatment groups included T₁ (proctodeal gland foam), T₂ (quail egg albumen), T₃ (Shit’s extenders), and T₄ (control group with undiluted semen). Each group consisted of five replicates with 15 birds per replicate. Additionally, 75 male breeder quails with an average weight of 170 ± 7 g were housed separately for semen collection and analysis. The female birds were housed in colony laying cages (60 × 60 × 24 cm) (Chelmońska et al., 2007; Arif et al., 2022), with a stocking density of 240 cm² per bird. They were 7 weeks old at the time of egg production, and were exposed to 16 hours of illumination during the laying periods, aided by artificial light (Arcadia 20 W Fluorescent Bird Lamp, 2-4% UVA; Arcadia Products plc, Redhill, UK). The Arcadia 20 W Fluorescent Bird Lamp typically produces a cool light designed to simulate natural daylight. These lamps usually have a color temperature of around 5500K to 6000K, closely resembling daylight. During the growing period, all treatment groups were given a quail grower diet (1-5 weeks) containing 22% crude protein and 2900 kcal/kg metabolizable energy (Table 1). From the sixth week onwards, a layer diet containing 18% crude protein and 2800 kcal/kg metabolizable energy was provided to the birds (Farooq et al., 2015; Ghayas et al., 2017). The breeder ration was provided to the flock at a rate of 30 g per day per bird (Table 1). The male birds were kept in cages (Ventury Welders cages measuring 22 × 20 × 15 cm) equipped with feeders and drinkers.

Table 1
Composition of the diet.

Ingredients (%)	Phases
	Grower	Production
Yellow corn	47.60	44.80
Wheat	15.32	20.50
Soybean meal (48%)	29.58	20.50
Protein concentrate	5.00	5.00
Fat	0.00	0.70
Calcium carbonate	1.00	7.00
Di-calcium phosphate	1.20	1.20
Salt	0.30	0.30
Total	100.00	100.00
Nutrient
Metabolizable energy (kcal/kg)	2900	2800
Crude protein (%)	22.00	18.00
Crude fiber (%)	3.72	3.18
Lysine (%)	1.15	0.92
Methionine (%)	0.47	0.47
Methionine + Cysteine (%)	0.81	0.70
Calcium (%)	0.90	3.03
Phosphorus (%)	0.51	0.51

To record daily variations in temperature (°C) and relative humidity (%), a wet and dry bulb hygrometer (Mason’s type, Zeal, England) was placed in the middle of the house. The house had dimensions of 6.10 × 6.10m (37.21 m²). Readings were taken at 6:00 AM and 6:00 PM. The minimum and maximum mean temperature and humidity ranged from 15.4 to 31.2°C, and 50 to 75%, respectively.

Extender’s composition

The Shit extender used in the study consisted of the following components per 100 mL of distilled water: 0.90 g of sodium chloride (NaCl), 0.10 g of sodium bicarbonate (NaHCO₃), 0.02 g of potassium chloride (KCl), 0.02 g of calcium chloride (CaCl₂), 0.01 g of magnesium chloride (MgCl₂), and 0.87 g of fructose. The pH of the extender was 7.3 (Shit et al., 2010).

Semen collection

Males were provided 14 hours of light. The timing of the collection was carefully planned to ensure that no more than 20 minutes passed between semen collection and insemination. A bird holder was used to collect the semen, allowing for easy manipulation of the males with free hands. To extract the frothy secretion from the cloacal gland, the left hand was used to apply pressure and force the secretion out, while the right hand cleared the secretion deposited at the vent using a clean towel. The right hand’s second finger was placed below the pubic bones and gentle upward pressure was applied. The thumb and index finger of the left hand applied lateral pressure to the cloacal region (Singh et al., 2011b; Wong et al., 2024). The semen, extruded from the vasa differentia, was drawn into a 30 mL sterile (10 × 75 mm) glass test tube (Fisher Scientific, Pittsburgh, Pennsylvania 15105, USA). To prevent sperm dehydration due to the small ejaculate volume, the semen from each male was immediately diluted two-fold with different semen extenders, and then the diluted samples were pooled (Blanco et al., 2009).

Semen extenders

One gram of freshly collected semen was cleansed of foam and then mixed with 1 mL of normal saline. The mixture was homogenized for 10 minutes by centrifugation at 3000 revolutions per minute. The resulting supernatant was used as 100% foam extract. To create a 5% foam extract, the extract was diluted in a ratio of 1:20 with normal saline. For insemination of the birds, a two-fold dilution of this extender was used, with 30 µL of diluted semen (Biswas et al., 2010).

Quail eggs albumen

The albumen from quail eggs was collected and homogenized. This was done by using a two-fold dilution of an extender, or by insemination using a 30 µL artificial insemination gun.

New semen diluent

A new semen diluent for quail semen was prepared using ingredients available in the local market. The diluent contained NaCl at a concentration of 0.90 g per 100 mL of distilled water, NaHCO₃ at a concentration of 0.10 g, KCl at a concentration of 0.20 g, CaCl₂ at a concentration of 0.02 g, MgCl₂ at a concentration of 0.01 g, and fructose at a concentration of 0.08 g. The pH of the diluent was 7.3. For the insemination of the birds, a two-fold dilution was prepared and 10 µL of semen was used (Shit et al., 2010).

Measurements

Post dilution semen parameters

After diluting the semen with the aforementioned semen extenders at a ratio of 1:2, the semen was evaluated for various parameters, including volume, motility, sperm concentration, live/dead sperm count, and morphological defects. The volume of the semen was measured by collecting it in a sterile glass test tube (10 × 75 mm, Fisher Scientific, Pittsburgh, Pennsylvania 15105, USA) with a capacity of 30 µL. To determine motility, fresh semen was diluted at a ratio of 1:200 (Tabatabaei et al., 2009), and the different semen extenders were used for different time intervals of 0 min, 30 min, 60 min, and 90 min (Shit et al., 2010). One drop of diluted semen was placed on a slide at a temperature of 27°C, and a cover slip was placed over it to ensure even distribution. The observation was conducted using a microscope with a magnification of 400x (Model N-400ME, CEL-TECH Diagnostics, Hamburg, Germany). Sperm concentration was determined using a hemocytometer (Levy chamber with double Neubauer ruling, Clay-Adams, A-2900). The entire semen sample was diluted in a spermicidal solution (Qureshi, 2011). A drop of diluted semen was placed on both ends of the hemocytometer, and the sperm count was calculated using the following formula:

$C=50000\times N\times D$

Where;

C = Concentration of spermatozoa per volume (mL)

N = number of spermatozoa

D = dilution rate

This observation was also performed using a microscope with a magnification of 400x (Model N-400ME, CEL-TECH Diagnostics, Hamburg, Germany).The live/dead sperm count was determined using smear staining with eosin-nigrosin, based on 300 spermatozoa (Tabatabaei et al., 2009; Qureshi, 2011). To perform the staining, the slide was pre-warmed to body temperature and a drop of eosin-nigrosin stain was placed on the slide. A small drop of semen was then added near the stain. The edge of another slide was placed on top of drop of stain and semen, and the two were mixed by sliding them back and forth a few times at 27°C. A second smear of stain was then prepared over the surface of the first one. The live/dead sperm count was determined by observing the stained slide under a microscope at 400x magnification (Model N-400ME, CEL-TECH Diagnostics, Hamburg, Germany). Live sperm appeared white, while dead sperm appeared red (Qureshi, 2011). Morphological defects were also assessed by observing 300 cells for each preparation, including defects in the head, mid-piece, tail, and other deformities (Tabatabaei et al., 2009).

Artificial insemination

The female quails were inseminated once a week between 9:00 and 10:00 AM, following egg-laying (Malecki & Martin, 2004). To perform artificial insemination, gentle pressure was applied to the left side of the abdomen to cause vaginal eversion. Once the vaginal eversion occurred, the AI gun (adjusted to 30 µL) loaded with diluted semen was inserted into the vagina. The pressure was then released, allowing the cloaca to return to its original position. Finally, the diluted semen was carefully delivered through the pipette, reaching a depth of 1 centimeter (cm) into the oviduct, following the established protocol (Shit et al., 2010).

Egg collection and incubation

Eggs from each of the four groups were collected separately, 3-4 times a day. They were then stored for three days at room temperature (18°C) with a relative humidity of 70-80%. Once all the eggs were gathered, they were set in the incubator (Circulated Air Hova-Bator Incubator, Model No. 1590, Savannah, GA, USA) for incubation. The eggs were incubated at the university poultry farm using an automatic incubator. The incubator maintained a temperature of 99.6°F (37.6°C) and a relative humidity of 50 to 60%. To ensure proper development, the eggs were manually turned 3-4 times a day at a 45° angle. After 15 days, all the eggs were transferred to a hatcher (Humidaire Incubator Company, New Madison, OH, Belgium) for hatching. In the hatcher, the eggs were kept at a temperature of 98°F (36.67°C) and a relative humidity of 75 to 80% (Seker et al., 2004).

The following egg’s parameters were studied:

Hatching traits

The percentage of egg fertility was calculated based on 20 eggs per replicate using the following formula;

$\%fertility=\left(Thenumberoffertileeggs\right)/\left(Totalnumberofeggslaid\right)\times 100$

Similarly, the percentage of egg hatchability was determined using the formula below:

$\%hatchability=\left(Thenumberofhatchedchicks\right)/\left(Numberoffertileeggs\right)\times 100$

Data analysis

The data collected were analyzed using the one-way analysis of variance technique. This was done through the general linear model procedure in the statistical package for social sciences (SPSS) software. To determine significant differences among treatments, Duncan’s multiple range test was used with a significance level of p≤0.05 (Duncan, 1955). The following mathematical model was applied:

$Yij=\mu +\tau i+ϵij$

Where;

Yij = observation of dependent variable recorded on the ith treatment

μ = Population mean; τi = Effect of ith treatment (i = 1, 2, 3, 4)

ϵij = Residual effect of jth observation in ith treatment NID ~ 0, σ 2.

RESULTS

Semen volume and sperm concentration

This study assessed various parameters of quail semen (Table 2). The average semen volume observed was 18.73 μL, with individual ejaculates ranging from 13 μL to 22 μL. Similarly, the concentration of spermatozoa varied among individuals, averaging 686 × 10⁶ sperm/mL, with a range from 580 × 10⁶ to 800 × 10⁶ sperm/mL.

Table 2
The impact of male quail breeders on semen volume, sperm concentration, and the presence of morphological defects.

No of males	SV (μmL)	SC (n×106/mL)	LNS (%)	Deformities in spermatozoa (%)
				MC	BN	MPC	OC
1	21.11	800.00	81.44	2.50	1.60	1.20	5.20
2	19.65	650.00	72.68	3.00	2.10	3.60	4.40
3	16.64	730.00	79.59	3.22	1.40	1.30	4.20
4	22.31	580.00	80.89	3.30	2.10	1.10	5.10
5	13.98	670.00	77.03	2.60	1.30	1.00	4.30
Mean	18.73	686.00	78.32	2.92	1.70	1.64	4.64

• SV: semen volume, SC: sperm concentration, LNS: live normal spermatozoa, MC: macrocephalic, BN: bent neck, MPC: mid-piece changes, OC: other changes.

Morphological defects

In the present study, the average percentage of morphologically normal spermatozoa per ejaculate was 78.32%, with a range from 72.68% to 81.44%. The observed deformities included macrocephalic, bent-neck, mid-piece, and other deformities. Macrocephalic deformities ranged from 2.50% to 3.30%, while bent-neck deformities varied from 1.30% to 2.10%. The average percentages for mid-piece and other deformities were 1.64% and 4.64%, respectively. The range for mid-piece deformities was 1.10 to 3.60%, and for other deformities it was 4.20% to 5.20%. Overall, the total deformities observed in all birds amounted to 10.90% (Table 2).

Live/dead sperm count and sperm motility

This study assessed the effects of various semen extenders on live sperm count and motility in quail semen (Table 3). The extenders tested included proctodeal gland foam (T₁), quail egg albumen (T₂), Shit’s extenders (T₃), and a control group with undiluted semen (T₄). Results indicated that the T₁ group (proctodeal gland foam) had the highest live sperm count, followed by T₂, T₃, and T₄. Additionally, Table 4 presents the sperm motility results for all four groups. The type of semen extender was found to significantly affect sperm motility, with the T₁ group demonstrating the highest motility, followed by T₂, T₃, and the control group (T₄).

Table 3
Effect of various semen extenders on live and dead sperm count at varying time intervals.

Effect1	0 (min)		30 (min)		60 (min)		90 (min)
	Live	Dead	Live	Dead	Live	Dead	Live	Dead
T1	92.88a± 0.39	7.10d± 0.39	84.55a± 0.86	15.44d± 0.86	79.66a± 0.69	20.33d± 0.68	67.77a± 0.48	32.22d± 0.47
T2	87.88b± 0.58	12.11c± 0.58	72.00b± 0.57	28.00c± 0.57	38.77b± 0.58	61.22c± 0.58	26.40b± 0.43	73.66c± 0.42
T3	81.33c± 0.57	18.66b± 0.57	60.00c± 0.57	40.00b± 0.57	26.40c± 0.43	74.90b± 0.42	10.33c± 0.53	89.33b± 0.53
T4	74.88d± 0.77	24.99a± 0.77	32.10d± 0.77	67.33a± 0.80	10.83d± 0.50	88.66a± 0.50	00.00d± 0.00	100.00a± 0.00

• ^a
  ^-d Means followed by different superscripts are statistically significant at alpha = 0.05 levels.
• ¹
  T₁ : Proctodeal gland foam extender, T₂ : Shit’s extender, T₃ : Quail eggs albumen extender, T₄ : control group.

Table 4
Effect of various semen extenders on sperm motility (%) at varying time intervals.

Effect1	0 (min)	30 (min)	60 (min)	90 (min)
T1	90.28a±1.02	85.16a±1.42	72.98a±0.95	66.29a±1.70
T2	85.42ab±.98	65.16b±1.92	15.35b±1.02	00.00b±0.00
T3	72.23b±1.14	55.58c±1.41	10.55b±1.02	00.00b±0.00
T4	74.46b±1.01	40.12d±0.61	00.00c±0.00	00.00b±0.00

• ^a
  ^-d Means followed by different superscripts are statistically significant.
• ¹
  T₁ : Proctodeal gland foam extender, T₂ : Shit’s extender, T₃ : Quail eggs albumen extender, T₄ : control group.

Hatching traits

Table 5 summarizes the fertility and hatchability outcomes for the four experimental groups: T₁ (proctodeal gland foam), T₂ (quail egg albumen), T₃ (Shit’s extenders), and T₄ (control group with undiluted semen). The findings revealed that the T₁ group exhibited the highest fertility rates, followed by T₂, T₄, and T₃. Similarly, hatchability was significantly higher in the T₁ group compared to the others.

Table 5
Effect of various semen extenders on quail egg fertility and hatchability.

Effect2	Parameters1
	F (%)	H (%)
T1	87.18a±2.21	62.28a±2.08
T2	74.03b±2.28	49.50b±3.27
T3	33.21d±1.36	48.07b±1.63
T4	51.01c±3.40	46.44b±1.68

• ^a
  ^-d Means followed by different superscripts are statistically significant.
• ¹
  F: fertility, H: hatchability.
• ²
  T₁ : Proctodeal gland foam extender, T₂ : Shit’s extender, T₃ : Quail eggs albumen extender, T₄ : control group.

DISCUSSION

Semen volume and sperm concentration

Semen production and volume in poultry are influenced by factors such as diet, age, and environmental conditions (Zaghari et al., 2011). In the present study, the average semen volume observed was 18.73 μL, with individual ejaculates ranging from 13 μL to 22 μL. The variations in semen volume among individual quails may be attributed to genetic differences, since diet, management, and temperature were kept consistent. These results are consistent with those of Chelmońska et al. (2008), who collected semen from different male quails and observed variations in volume based on factors such as cloacal gland size, age of the males, collection procedure, and frequency of collection. Similar findings were also reported by another study (Shit et al., 2010). In their experiment, Qasimi et al. (2017) collected semen from quails with different sizes of cloacal glands and discovered that cloacal gland size significantly influenced the volume and concentration of quail spermatozoa. In contrast to our findings, Wentworth & Millen (1963) reported an average volume of 10 µL per ejaculate, which was lower than what we observed. This difference in volume may be attributed to the use of a different method for semen collection.

Sperm concentration is closely linked to testicular health and the efficiency of spermatogenesis (Aire, 2007; Santiago-Moreno et al., 2016). These processes are regulated by testosterone and other gonadotropins, such as luteinizing hormone and follicle-stimulating hormone, which help maintain a high density of spermatozoa (Sengupta & Elbardisi, 2019). Quails with well-developed testes and an optimal hormonal balance typically exhibit higher sperm concentration levels (Parkhurst et al., 1998).The sperm concentration of the quail semen collected in this study ranged from 580 to 800 × 10⁶ per mL, which is similar to the results reported by Chelmońska et al. (2008). In their experiment, they collected semen from different individual quails and found a spermatozoa concentration ranging from 592 to 800 × 10⁶ per mL. However, the sperm concentration in our trial was higher than that reported by Buxtton & Orcut (1975) (469 × 10⁶ per mL), Fujihara & Koga (1991) (42.92 × 10⁶ per mL), and Bunaciu et al. (1994) (220-230 × 10⁶ per mL). This difference could be attributed to variations in age or the method of semen collection. In the experiment by Chelmońska et al. (2006), the highest spermatozoa concentration (2240-2640 × 10⁶ per mL) was reported, which could be due to breed differences in their study.

Morphological defects

Sperm morphology is affected by spermiogenesis (Biagi et al., 2016) and by processes that occur after spermiation (Khalil et al., 2019). Inexperienced handling of semen samples can lead to issues during cooling and freezing, which may result in acrosomal damage and tail abnormalities (El-Bahrawy et al., 2017). Abnormal sperm shapes may include head defects like microcephaly and macrocephaly, as well as mid-piece defects such as proximal cytoplasmic droplets, distal mid-piece reflexes, and segmental aplasia of the mitochondrial sheath (Gruhot et al., 2019).

The findings of this study indicated that the average percentage of morphologically normal spermatozoa per ejaculate was 78.32%, with deformities ranging from 72.68 to 81.44% across individual birds. Observed abnormalities included macrocephalic spermatozoa, bent-neck deformities, mid-piece abnormalities, and other defects. The reported range (72.68-81.44%) reflects individual variation within the population. Overall, the total deformities for all birds were 10.90%, which is consistent with the findings of Chelmońska et al. (2008), who observed an average of 11% defects in different birds. Our results also align with the findings of Bunaciu et al. (1994), who specifically studied sperm head deformities and reported a percentage ranging from 4.05% to 6.63%. In a study by Fujihara et al. (1989), the morphological defects of quail spermatozoa were examined at different time intervals, and it was found that after 24 hours of storage, 70% of the defects observed were broken necks, similarly to what is observed in poultry semen.

Live/dead sperm count and sperm motility

Sperm viability is crucial for motility and the ability to fertilize. As sperm viability declines, their potential to achieve fertilization is reduced (Kumaresan et al., 2017; Baiee et al., 2018). In the present study, different semen extenders had a significant impact on the live/dead sperm count, as shown in Table 3. T₁, which utilized proctodeal gland foam, had the highest live sperm count in both fresh and storage conditions. This can be attributed to the foam’s biochemical properties, which closely mimic the natural reproductive environment of birds. As a secretion from specialized male bird glands, proctodeal foam provides an ideal medium for sperm preservation (Abuoghaba et al., 2024). Its antioxidant content neutralizes reactive oxygen species, protecting sperm membranes from oxidative damage (Kaltsas, 2023). Additionally, the foam’s lipid content serves as a readily available energy source, supporting ATP production that is essential for sperm survival and motility (Islam et al., 2021).

These findings are consistent with Chelmońska et al. (2008), who observed 94.60% live spermatozoa in quail semen. Similarly, Biswas et al. (2010) conducted research on different concentrations of foam as quail semen extenders and found a higher live sperm count at 5% concentration. Our results also align with those of Shit et al. (2010), who reported similar findings with Shit’s extender. However, our findings for the control group contradict the results of Chelmońska et al. (2008), who reported a higher live sperm count in their research. This difference may be attributed to variations in environmental temperature or storage conditions.

Sperm motility is a key indicator of male fertility potential (Bergeron & Manjunath, 2006). To achieve optimal fertility, sperm cells must maintain high motility levels (Martínez‐Pastor et al., 2010; Amann & Waberski, 2014). Poor motility or the presence of immotile sperm are significant indicators of male infertility (Longobardi et al., 2017). In the present study, semen extenders had a significant impact on sperm motility. Group T₁, which utilized proctodeal gland foam, had the highest number of motile spermatozoa. This high motility in group T₁ may be attributed to the lipids in the foam, which provide a readily available energy source, supporting motility through ATP production (Farooq et al., 2015). Additionally, the antioxidants present in the foam help preserve the axoneme, the structural core of sperm motility, from oxidative damage (Kowalczyk, 2022). Biswas et al. (2010) conducted a study on different foam concentrations and their effect on quail sperm motility. They reported that a 5% foam concentration significantly affected sperm motility under both fresh and storage conditions, which aligns with our own research findings. Similarly, Shit et al. (2010) examined different semen extenders at various time intervals in quails and obtained comparable results. The findings of Chelmońska et al. (2008) were also consistent with our own research.

Hatching traits

The primary objective of poultry breeding operations is to produce healthy and viable chicks. Since only fertile eggs can hatch into chicks, egg fertility is a crucial factor. Low fertility or infertility can significantly impact the productivity of breeder flocks, leading to economic challenges. Since both male and female birds contribute equally to egg fertility, a decline in fertility may arise from factors related to either or both sexes (Farooq, 2014).

Group T₁, which utilized proctodeal gland foam, exhibited the highest fertility rates, likely due to its unique biological composition and compatibility with the avian reproductive system. This foam closely mimics the natural secretions of male birds, providing an ideal medium for sperm storage, transport, and survival. It contains bioactive components such as lipids, proteins, and antioxidants that enhance sperm viability (Agarwal et al., 2016). In vitro studies have shown that quail sperm tend to cluster without foam (Kobayashi et al., 1972; Ogawa et al., 1974). In contrast, when foam is added, they disperse effectively in the medium, sustain motility for longer durations, and exhibit high metabolic activity (Fujihara et al., 1989; Chelmońska et al., 2006; Biswas et al., 2010; Singh et al., 2011a), which may positively impact fertility. Our findings support this, as fertility rates in the T₁ group were the highest among the experimental groups. Cheng et al. (1989) found that, when stored with foam at room temperature, sperm maintain motility for 90 minutes, whereas motility is lost within 10 minutes without foam. Similarly, our results align with those of Fujihara & Koga (1991), who found that quails inseminated with foam-mixed semen had higher fertility rates than those inseminated with semen alone. Furthermore, Chelmońska et al. (2008) achieved comparable fertility using undiluted fresh semen and diluted semen with a proctodeal gland foam extender in quails. Shit et al. (2010) also reported similar fertility rates when using Shit’s extender. Similarly, Farooq et al. (2015) found that proctodeal gland foam enhances fertilization by improving the motility of the sperm. However, our results differ from the findings of Chelmońska et al. (2006), who reported higher fertility with fresh undiluted semen and lower fertility with diluted semen, possibly due to the type of extender or the high dilution rate used in their study.

Hatchability is a key economic trait in the poultry industry due to its significant impact on chick production (Wolc et al., 2010). In the current trial, group T₁ demonstrated significantly higher hatchability compared to the other groups. This increased hatchability in group T₁ may be attributed to the high fertility observed in the group, as only fertile eggs can successfully hatch into chicks. In a study by Chelmońska et al. (2006), a hatchability of 69% was reported with the use of a foam extender, which is higher than what we observed in our experiment. The lower hatchability in our study may be due to a lower frequency of insemination, as they conducted it twice and thrice per week. Similarly, Shit et al. (2010) reported findings that are comparable to our research.

CONCLUSION

These findings suggest that using proctodeal gland foam as a semen extender in Japanese quails may enhance hatching traits. The positive results with this extender also support further studies on quail semen storage and cryopreservation. However, the research on the effect of various semen extenders on the reproductive performance of Japanese quail has certain limitations. Firstly, due to species-specific reproductive biology, the findings may not be widely applicable to other poultry species. Secondly, the effectiveness of semen extenders may vary, since factors such as extender composition, storage conditions, and quail genetics can influence the outcomes.

ACKNOWLEDGEMENTS

The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSPD2025R694), King Saud University,Riyadh, Saudi Arabia, and the faculty staff of the Poultry Science department at the University of Agriculture Peshawar, Pakistan, for their assistance in conducting the trial.

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  Tatiana Carlesso dos Santos

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Publication Dates

• Publication in this collection
  09 May 2025
• Date of issue
  2025

History

• Received
  21 Apr 2024
• Accepted
  17 Feb 2025