Productivity in broiler breeder operations is largely determined by reproductive efficiency since male fertility directly affects fertilization success, hatchability rates, and overall flock performance (Ncho et al., 2025). Roosters’ fertility depends mainly on semen quality, which is defined by sperm concentration, motility, viability, and volume. Maximizing fertilization rates depends on maintaining good semen quality, especially in industrial chicken farming systems where natural mating is still the major technique of reproduction (Wang et al., 2024). Although environmental variables, diet, and genetic selection affect semen quality, two key factors that can substantially influence reproductive function are age and body weight (Tadondjou et al., 2013; Gonçalves et al., 2015; Ayeneshet et al., 2024).

Age-related changes in semen traits have been recorded in broiler breeder males; older roosters often show better sperm motility because of the development of their reproductive system and hormonal stability (Barbarestani et al., 2025). Advancing age, however, can also lead to oxidative stress and cellular damage, which may reduce sperm viability and overall semen quality (Haryuni et al., 2022). Furthermore, body weight is a critical factor for reproductive performance since both underweight and overweight states can impair sperm production and function. Underweight rooster may have nutritional deficiencies that affect spermatogenesis, therefore lowering sperm count and compromising semen quality (Shaheen et al., 2023). On the other hand, overweight roosters are sometimes associated with increased fat deposition, hormonal imbalances, and decreased testicular efficiency, which can adversely influence sperm motility and fertility outcomes (Olubowale, 2013).

The combined effects of age and body weight on semen quality in male broiler breeders are still not well understood, despite these findings. Previous research has focused on the individual effects of age or weight, although understanding their interaction is vital for creating effective management strategies to improve reproductive success. Inadequate weight control in breeding males can lead to suboptimal semen production, thereby reducing the fertilization success and overall flock fertility (Oke et al., 2024). Therefore, examining the interaction between age and body weight in broiler breeder males is required to identify the ideal weight range and age for optimizing semen quality and fertility potential. Although prior research has examined the separate effects of age and body weight, there is limited information regarding their interaction in tropical environments, particularly for Ross 308 breeder males. This study offers new insights into the interactions between age and weight on semen characteristics under equatorial management systems.

Notwithstanding the available evidence regarding the separate impacts of age and body weight on semen characteristics, there is still limited information regarding their synergistic effects in broiler breeder males, especially in commercial strains such as Ross 308. Addressing this issue is essential for enhancing male reproductive efficiency through optimized management strategies. This study aimed to evaluate the interactive effects of age and body weight on semen quality parameters in Ross 308 broiler breeder males. The evaluated parameters included sperm viability, sperm motility, sperm concentration, and semen volume. The findings are expected to support the identification of optimal age–weight combinations that maximize fertility potential and semen quality. It is hypothesized that semen characteristics are significantly influenced by both age and body weight, as well as by their interaction. Specifically, roosters with normal body weight at older ages will demonstrate superior semen quality and sperm motility compared with other age–weight combinations.

The study was conducted at the Faculty of Sustainable Agriculture, Sandakan, Sabah, Malaysia (5°56' north latitude 118°00’ east longitude, with altitude of 25 m with a predominant tropical rainforest climate climate).

After approval by the Universiti Malaysia Sabah Animal Ethics Committee (JEHUMS), experimental procedures were implemented in accordance with the Researchers’ Guidelines on the Code of Practice for the Care and Use of Animals for Scientific Purposes (Approval No. AEC 0012/2024).

Twenty-four Ross 308 broiler breeder males were randomly assigned to six experimental groups, each consisting of four roosters, and were confined in wire-floored metal cages (65 × 55 × 70 cm), individually placed in a naturally ventilated poultry house. The facility was constructed to allow for adequate airflow and exposure to ambient environmental conditions, including natural daylight. Birds were managed under a natural photoperiod without artificial lighting, with day length ranging approximately from 12 to 13 h throughout the study period. The experimental treatments comprised two age groups (25-29 weeks and 30-39 weeks) and three weight classifications: normal (4.0-4.5 kg), underweight (3.0-3.5 kg), and overweight (5.0-5.5 kg). Body-weight classifications were determined based on the Ross 308 Performance Objectives manual Ross 308 FF Parent Stock: Performance Objectives (Aviagen, 2011).

The roosters received commercial male broiler breeder pellets containing 13% moisture, 13% crude protein, 3.5% crude fat, 7% crude fiber, 4% calcium, and 0.7% phosphorus. Feed and water were provided ad libitum. Each rooster was provided with approximately 110-120 g of feed daily, adjusted according to its respective body weight group. Feed was provided once daily at 06:00 h.

Prior to data collection, all birds underwent a two-week acclimatization period, during which they were conditioned for semen collection using the abdominal massage method (Burrows and Quinn, 1937). Semen was obtained from each rooster, and individual sperm evaluation were performed. Health status and general behavior was monitored twice daily throughout the study. Cage trays were cleaned weekly, and accumulated waste was removed daily to maintain hygiene.

Volume was measured immediately after semen collection using a 1 mL syringe. The evaluation of color was conducted using the color score, 1 - Watery, 2 - Cloudy and 3 - Creamy (Mohamad Asrol and Abdul Rashid, 2017).

The sperm concentration of an output was assessed using a hemocytometer chamber following a dilution with distilled water at a 1:400 ratio, expressed as ×10⁹ mL⁻¹.

A drop of undiluted semen was applied to a microscope slide without a coverslip, and mass motility was evaluated by examining it under a compound microscope at 100× magnification. A scale of 1 to 5, based on Pimprasert et al. (2023) was used to assess motility.

According to recognized protocols, semen was diluted using a modified Ringer’s solution at a 1:100 ratio (semen:extender) for progressive motility (Mohamad Asrol and Abdul Rashid, 2017). A drop of diluted semen was examined under a microscope at 400× magnification after being placed on a slide and covered with a glass coverslip. The proportion of motile spermatozoa exhibiting moderate to rapid progressive movement was utilized to assess motility. Each sample was analyzed in at least three microscopic fields.

The eosin-nigrosin stain method was used to evaluate the sperm viability (Mohamad Asrol and Abdul Rashid, 2017). An equal volume of eosin-nigrosin stain was well mixed with a 10 µL sample of semen. The mixture was then spread uniformly on a microscope slide and allowed to air-dry. A phase-contrast microscope at 400× magnification was used to view the slide. A total of 200 spermatozoa per sample were evaluated. Unstained sperm were classified as viable, whereas stained sperm were regarded as nonviable.

Data were analyzed using SAS^® OnDemand for Academics version 9.4 statistical software (SAS Institute Inc., 2024). Initially, the data were assessed for normality and homogeneity of variance, followed by analysis using PROC GLM under a completely randomized design (CRD). The statistical model included the main effects of age and body weight, as well as their interaction (age × body weight). When a significant interaction was detected, simple-effect comparisons were performed using Tukey’s post-hoc test to examine differences within age or body weight classes. Differences were deemed significant when P<0.05. The impact of age and weight was assessed using the following model:

in which Yijk is the quantitative response variable, μ is the overall mean, αi is the fixed effect of age, βj is the fixed effect of weight, (α×β)ij is the interaction effect, and eijk is the random error.

The semen characteristics of Ross 308 broiler breeder males were significantly influenced by the interaction between age and body weight (Table 1).

Table 1

	25-29 weeks			30-39 weeks			SEM	P-value
	Normal	Underweight	Overweight	Normal	Underweight	Overweight		Age × Weight
Colour (score)	3.00	2.71	2.50	1.78	2.33	3.00	0.72	0.27
Volume (mL)	1.55ab	0.60a	0.70a	0.10a	0.85ab	2.35b	0.51	0.04
Concentration (109/mL)	1.35b	0.57ab	0.74ab	0.22a	0.7ab	1.13ab	0.38	0.07
Gross motility (score)	4.50	2.17	2.92	4.50	3.50	4.80	1.04	0.37
Individual motility (%)	88.00a	31.67c	27.50c	80.00a	56.67b	84.00a	5.97	0.02
Live (%)	57.70	49.67	56.75	66.00	68.09	64.00	10.50	0.72
Dead (%)	41.40	45.54	42.75	33.50	31.34	35.10	9.09	0.85

Means followed by different letters in the same row differ from each other (P≤0.05) by the Tukey’s test.

The interaction between age and body weight had a significant impact on semen volume and individual sperm motility (P<0.05), while age had no significant effect on semen color, sperm concentration, gross motility, or sperm viability (P>0.05). The significant interaction indicates that the influence of body weight on motility varies with age. Normal-weight roosters exhibited the highest motility at 25-29 weeks, whereas older birds (30-39 weeks) maintained higher motility even at greater body weights.

At 25-29 weeks, normal-weight roosters produced the highest semen volume (1.55 mL), whereas underweight and overweight birds had lower volumes. However, at 30-39 weeks, overweight roosters showed a significant increase in semen volume (2.35 mL), while normal-weight and underweight birds produced significantly lower volumes (0.10 mL and 0.85 mL, respectively). Overweight roosters at 30-39 weeks had a 51.6% increase in semen volume compared with their volume at 25-29 weeks. In contrast, semen volume in normal-weight roosters decreased by 93.5%, from 1.55 mL to 0.10 mL, with age progression.

A significant interaction effect was also observed for individual sperm motility (P<0.05). At 25-29 weeks, normal-weight roosters exhibited the highest motility (88.00%), whereas both underweight (31.67%) and overweight (27.50%) birds had significantly lower values. This represents a 64.0% and 68.8% reduction, respectively, compared with normal-weight roosters at the same age. By 30-39 weeks, overweight and normal-weight roosters showed the highest motility (84.00% and 80.00%, respectively), while underweight birds continued to display lower motility (56.67%). Motility in underweight birds was 32.6% lower than that of overweight birds within the same age group.

Although sperm viability and mortality were not significantly influenced by the interaction (P>0.05), the percentage of live spermatozoa was slightly higher in normal-weight and underweight roosters at 30-39 weeks (66.00% and 68.09%, respectively).

Individual sperm motility was significantly higher in older roosters (30-39 weeks) (69.33%) than in younger roosters (25-29 weeks) (41.26%) (P<0.05) (Table 2). This represents a 67.9% increase in motility with advancing age.

Table 2

	25-29 weeks	30-39 weeks	SEM	P-value
Colour (score)	2.74	2.29	0.52	0.38
Volume (mL)	0.85	0.83	0.26	0.67
Concentration (109/mL)	0.89	0.61	0.15	0.42
Gross motility (score)	3.96	3.19	1.08	0.17
Individual motility (%)	41.26b	69.33a	35.64	0.01
Live (%)	66.03	54.71	110.18	0.11
Dead (%)	43.23	33.31	82.63	0.11

Means followed by different letters in the same row differ from each other (P≤0.05) by the Tukey’s test.

Age did not significantly influence other semen parameters, such as semen color, volume, sperm concentration, gross motility, and viability (P>0.05). However, younger roosters showed a higher percentage of live sperm (66.03%) than older birds (54.71%).

Semen parameters, including sperm motility, were strongly impacted by body weight (P<0.05). Normal-weight roosters showed the highest motility (84.00%), while underweight and overweight had significantly reduced motility (44.17% and 46.33%, respectively) (Table 3). Motility in underweight roosters was reduced by 47.4%, and in overweight roosters by 44.9%, compared with normal-weight roosters.

Table 3

	Normal	Underweight	Overweight	SEM	P-value
Colour (score)	1.25	2.48	2.75	0.52	0.76
Volume (mL)	0.58	0.75	1.25	0.26	0.20
Concentration (109/mL)	0.79	0.64	0.87	0.15	0.60
Gross motility (score)	4.50	3.27	3.87	1.08	0.34
Individual motility (%)	84.00a	44.17b	46.33b	35.63	0.01
Live (%)	61.85	58.8	60.37	110.17	0.92
Dead (%)	37.45	38.44	38.94	82.63	0.97

Means followed by different letters in the same row differ from each other (P≤0.05) by the Tukey’s test.

Body weight did not alter semen color, volume, sperm concentration, mass motility, viability, or mortality (P>0.05). Overweight roosters had a semen volume of 1.25 mL, whereas normal-weight birds had 0.58 mL, although the difference was not significant (P>0.05). Sperm concentration was not significantly different across groups, with overweight roosters showing slightly higher concentrations (0.87 × 10⁹ sperm/mL) than normal-weight (0.79 × 10⁹ sperm/mL) and underweight birds (0.64 × 10⁹ sperm/mL).

This study illustrates that the quality of semen in Ross 308 broiler breeder males is substantially influenced by both age and body weight, with a particular emphasis on sperm motility. Older birds generally demonstrated enhanced motility, irrespective of their weight, while normal-weight roosters demonstrated superior motility compared with underweight and overweight birds. These results underscore the significance of age- and weight-specific management strategies to optimize reproductive performance in broiler breeders. Nevertheless, the study is constrained by the absence of hormonal and seasonal profiling, as well as the relatively small sample size, which may also affect semen characteristics. These factors should be considered in future research to enhance and generalize the results.