1. Introduction
Although the poultry sector contributes significantly to many national economies, especially developing countries, reduces unemployment, and provides high-quality proteins, its potential has been limited by several constraints, especially concerning genetic strains and reproductive capability for the production of quality day-old chicks. The use of birds with poor genetic potentials, use of substandard feeds and quality [1], pest and disease challenges [2], and poor management [3] have led to huge economic losses in countries with low or no regulations in the poultry industry. Other constraints are finance affecting investment in standard housing [4], decreasing hatchability, male infertility, and the supply of poor-quality day-old chicks (DOCs) [5]. The supply of quality day-old chicks is important because it is the backbone of any poultry production sector and it determines the success of poultry production, whether small or large scale [6]. Both fertility and hatchability are factors that determine day-old chick supply. A decline in the fertility of flocks is attributable to many factors [7–11], with the male factor accounting for approximately 50% of the genetic contribution in poultry production [6, 12]. Hence, male fertility is a major factor to be considered in the poultry industry but has received less attention because it is assumed that the male contributes nothing apart from the genetic material (DNA).
Male fertility is highly dependent on semen quality and other factors such as genetics and environment. Semen quality affects the fertility potentials of domestic fowls [13–16], and sperm quality is a principal predictor of its fertilizing potency [17]. Several sperm quality predictors have been found to influence fertility. The influences of sperm morphology, sperm motility, and sperm viability on sperm quality and fertility are widely reported [18–20]. The selection of roosters based on semen quality traits that may improve fertility has been suggested due to limited correlations between semen quality traits and fertility, which impedes the selection of effective parameters for roosters [13]. There is a study that confirms the positive effect of genetic strain, flock age, the weight of cock, and the breed of cock on sperm quality and fertility [21]. Peters et al. [22] discovered a positive correlation between breed and fertility. Nutrition has also been found to influence fertility [8]. Therefore, nutritional factors that could improve the fertility of poultry males and associated mechanisms to increase semen volume, sperm concentration, viability, forward motility, and polyunsaturated fatty acids in sperm, as well as protecting against oxidative damage, could help to optimize the sperm membrane functionality, mitochondrial activity, and sperm–egg penetration, and thus fertility enhances poultry productivity, especially in aging poultry males and heat-stressed environment [6, 8]. The objective of the current study was to assess the DNA fragmentation difference in two indigenous tropical breeds and their physical sperm quality difference as indicators of breeder male fertility potential.
2. Materials and methods
2.1. Experimental sites
The research was conducted at the Department of Animal Science, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ghana, which lies between 06°41″ N and longitude 01°33″ W at an elevation of 261 m above sea level. The average ambient temperature range is 23–31°C. All experiments were conducted according to the Procedure for Animal Research Ethics Committee (AREC) of the Kwame Nkrumah University of Science and Technology, Kumasi-Ghana, Quality Assurance, and Planning Unit (KNUST POLICY 0016) [23].
2.2. Experimental design and data collection
The birds for this study were obtained from the Department of Animal Science, KNUST. The selected cockerels comprised two different breeds. One of the breeds is a hybrid following a cross between the Lohmann brown commercial females and Rhode Island Red males with full feather cover (normal feathered [NF] birds) [24, 25]. The other breed was a naked neck (NN) chicken selected over six generations of backcrossing between the Lohmann brown commercial females and brown NN male birds [26]. The breeding males were permitted a week of acclimatization preceding the inception of semen collections. In addition, they were primed for semen collection via the abdominal massage procedure. The fowls were supplied with layer mash at 118 g/per/bird containing 180 g/kg of crude protein and 11.3 MJ ME. Water was given ad libitum.
Semen samples were collected from 10 males each from the two breeds by abdominal massage method after the cockerels have been trained for two days. The birds were managed intensively in a cage (1 m × 1 m × 1 m). Semen was collected into 1.5-ml calibrated Eppendorf tubes and allowed to liquefy (15 minutes) while they also cool to room temperature before being transferred into a Styrofoam box containing ice (4°C) to reduce the occurrence of a cold temperature shock to the sperms. Semen volume was measured, and semen viscosity was determined using scoring from 1 to 5 by visual examination. Semen samples were diluted with phosphate buffer saline in a ratio of 1:10 and a sample of each semen placed on a hemocytometer. The samples were viewed under a microscope at ×40 magnification to determine sperm concentration and sperm cells movement (sperm motility). Samples of the diluted semen were also mixed with trypan blue dye in 1.5-ml tube and viewed under the microscope at ×100 oil immersion to determine sperm morphology and sperm viability (total number of live sperms divided by the total number of sperms counted inside the hemocytometer grids and multiplied by 100). Like the sperm viability test, a trypan blue stain was used to identify normal versus abnormal sperm cells. Viable sperms that remained undeviating from the head to the tail were adjudged to have normal sperm cells, and abnormal sperm cells involved spermatids and spermatozoa with twisted necks, spermatozoa with head distortion, and some with missing tails [27].
Sperm mobility (percentages of progressive, non-progressive, and immotile sperm cells) was measured by observing a drop of the diluted ejaculate on a slide concealed with a glass cover under a microscope at ×400 resolution. Motility was evaluated on the proportion of sperm displaying forward movement or with a reasonable amount of quick progressive forward motion in enormous rings, whereas non-progressive sperms were those with little to adequate progressive movement on the spot convulsing motion. The immotile sperms were the non-moving spermatozoa [28]. A sample of semen was spun down to concentrate the sperm. The levels of sperm DNA fragmentation were determined by extracting DNA from the sperm using the phenol and chloroform methods. The resulting DNA was separated using gel electrophoresis along with a standard DNA marker. The separated DNA bands were viewed under ultraviolet light [29].
2.3. Statistical analysis
Data on semen volume and viscosity were analyzed using one-way analysis of variance with Proc. GLM procedure of SAS software version 9.4 [30]. The means were separated using the Duncan’s multiple range test at p < 0.05. Sperm quality characteristics were expressed as a percentage of the average total cells counted for each of 10 birds with samples pooled together for each breed.
3. Results and discussion
3.1. Physical characteristics of semen and sperm
The results show that the average semen volume was significantly higher in the NF birds compared to the NN birds (0.40 ml versus 0.24 ml; p = 0.0055). On the contrary, the semen was thicker as shown by the higher average viscosity score in the NN birds compared to the NF birds (3.9 versus 2.4) (Table 1).
The results also show that sperm concentration was higher in NF birds (4.3 × 106 sperms/ml) compared to the NN birds (3.0 × 106 sperms/ml) (Table 2). However, all other semen characteristics appeared lower in the sperms of NF birds compared to the NN birds. For sperm viability, the percentage of live sperm was 67% in NN birds as compared to NF birds which had 63%. On the other hand, the percentage of dead sperms was 37% versus 33% in NF versus NN birds. Further examination showed that there was a slightly higher percentage of normal sperm morphology in NN birds (71%) than in NF birds (69%). The abnormal sperms were 31% versus 29% in NF birds versus NN birds. There were higher percentages of progressive sperms (54%), lower non-progressive sperms, and lower immotile sperms (19%) in NN birds compared to NF birds which had lower progressive sperms, higher non-progressive, and immotile sperms (45%, 34%, and 21%), respectively.
Table 1
Differences in semen volume and viscosity between brown naked neck chickens and normal feathered brown chicken breeds in tropical weather
| Sperm Characteristics | Normal Feathered Birds | Naked Neck Birds | SEM | P Value |
|---|---|---|---|---|
| Semen volume (ml) | 0.399 | 0.237 | 0.036 | 0.0055 |
| Semen viscosity1 | 2.4 | 4.0 | 0.251 | 0.0005 |
Table 2
Differential characteristics of rooster sperms analyzed in two tropical breeds selected to reduce heat stress for increased fertility and hatchability
The results on semen volumes are consistent with the previous outcome by Peters et al. [22] and Ajayi et al. [31] that described that the semen volume of the NF chicken was greater (0.58–0.83 ml) than that of the NN birds (0.18–0.37 ml) in Nigerian local birds. The outcomes are different from Galal [32] who recorded higher semen volume in NN chickens compared to NF Dandarawi and Fayoumi Egyptian male chickens. Semen volume is positively correlated with sperm concentration and therefore chickens producing high volumes of semen have better sperm quality than those producing a low semen volume [33]. The NN chickens may therefore need further genetic programs targeting reproductive traits. It is not clear if birds producing higher volumes of semen will always have lower viscosity. The components of semen that could lead to higher viscosity may include the pH and sperm count. Viscosity is reported to make sperm movement difficult and as such it reduces sperm motility affecting fertility [34].
In humans, semen hyperviscosity (SHV), which has a prevalence of between 12% and 29%, can cause male infertility both in vivo and in vitro. Semen is made up of fluids that are released by male accessory glands and contains proteins that are necessary for semen to liquefy and coagulate [35]. Unusual seminal fluid viscosity is brought on by hypofunction of the prostate or seminal vesicles. The development of SHV may also be influenced by infection and increased seminal leukocyte counts. Additionally, oxidative stress and physiological and genetic variables may be involved in this seminal abnormality. A decrease in sperm count might result from hyperviscosity, which can hinder proper sperm motility in the female reproductive system [35].
The lower semen volume, lower sperm concentration, and high semen viscosity which are linked to poor reproductive ability are a challenge when selecting indigenous breeds for improvement. The reproductive traits are normally not a target for their selection, leading to poor semen indices and reproductive ability [33]. Similar results are established in 40–44-week-old indigenous Beijing-You breeder roosters having high and low sperm motility phenotypes such as semen characteristics, reproductive hormone concentrations, testicular histomorphology, fertility capacity, and genes expression of hematopoietic prostaglandin D synthase. The results showed that in roosters with similar body weight, the low sperm motility roosters had smaller testis mass, seminiferous tubules diameter, spermatogenic epithelium height, and Johnsen score than roosters with high sperm motility [35, 36]. Likewise, in Rhode Island Red chicken roosters, which are more indigenous than White Leghorn roosters, the semen volume, sperm motility, and viability were higher than in the latter [13]. In a comparison of semen quality between five strains, including two exotic breeds (Isa brown and Harco) and fizzled chickens, the NN cocks had the highest semen volume of 0.5 ml while NF had the least of 0.20 ml [37]. In the current study, the higher better sperm quality characteristics observed in the NN cocks could explain potential performance in higher temperatures based on the objectives of their selection, which includes a selection for resistance to heat stress and increased reproductive efficiency [26]. The study results agree with previous results, indicating that selection of roosters based on semen quality traits for improved fertility and multivariate analysis may help in the precise selection of breeds by comprehensive usage of different measures of sperm quality [13].
3.2. Molecular characteristics of sperm quality
Individual gel electrophoresis of DNA extracted from sperms obtained from the roosters examined showed a faster movement of DNA fragment on the agarose gel from the NF birds compared to NN birds. DNA samples from NN chicken sperms were found to be heavier in weight and were slower in their migration through the gel than those from the NF birds, except for samples 1 and 6 (Figure 1). All the DNA fragments from the NF rooster sperms were smaller and moved faster on the agarose gel and showed fragment sizes between 100 and 600 bp. The small fragment sizes are evidence of DNA fragmentation in the harvested semen in each of the breeds. The higher the DNA fragmentation, the lower the DNA fragment size [29]. The increased DNA fragmentation of the sperms of the NF may be due to heat stress unlike the NN chickens, which were observed to be more tolerant to high ambient temperatures (26–33°C). This could be due to the inherent nature of the NN birds or the presence of and adapting to heat stress [26]. It is well established that sperm DNA fragmentation correlates with male reproductive outcomes. Spanò et al. [36–39] reported lower fertility in males with a high level of sperm DNA damage. Sperm DNA fragmentation has been identified to affect certain sperm quality predictors such as sperm cell concentration [40] and sperm viability [41]. Sperm DNA fragmentation was found to have a strong correlation with lower sperm viability [42].
Figure 1
Gel electrophoresis and analysis of DNA extracted from sperms of naked neck chickens and normal feathered chickens (nn, naked neck; nf, normal feathered; L, 1,000 bp standard marker). Smaller fragment sizes were consistent with the product size expected from cells undergoing DNA degradation as a result of cell death which results from increased apoptosis due to stressful environmental conditions such as cold or heat stress [23, 29].
4. Conclusion
Our results demonstrate the potential for greater fertility ability in NN birds compared to normal neck feathered birds based on conventional semen analysis procedures and sperm DNA fragmentation studies. This underscores the importance of semen quality assessment as a tool or guide for selection of indigenous birds for breeding purposes for improved poultry production, especially in a hot tropical environment.
