Abstract
Background and Aim
The impact of exogenous melatonin on the sperm quality of small ruminants is controversial. Therefore, this study aimed to synthesize previous findings on the influence of melatonin injection on sperm quality, steroid hormones, and testicular blood flow in small ruminants.
Materials and Methods
Thirty studies were analyzed by computing the raw mean difference (RMD) as the effect size between the control and melatonin treatment groups, using the inverse of the variance for the random-effect model of the method of moments by DerSimonian and Laird. We assessed heterogeneity among studies using Q test. I2 statistic was used to classify the observed heterogeneity. We used Egger’s regression method to indicate publication bias.
Results
Melatonin injection (p < 0.05) affected sperm concentration (RMD = 0.42 × 109/mL), morphology (RMD = 2.82%), viability (RMD = 2.83%), acrosome integrity (RMD = 4.26%), and DNA integrity (RMD = 1.09%). Total motility (RMD = 5.62%), progressive motility (RMD = 7.90%), acrosome integrity (RMD = 8.68%), and DNA integrity (RMD = 2.01%) of post-thawed semen in the melatonin-treated group were also increased (p < 0.05). Similarly, treatment with melatonin (p < 0.05) enhanced total motility (RMD = 5.78%), progressive motility (RMD = 5.28%), curvilinear velocity (RMD = 4.09 μm/s), straight-line velocity (RMD = 5.61 μm/s), and average path velocity (RMD = 4.94 μm/s). Testosterone (RMD = 1.02 ng/mL) and estradiol 17-ß levels (RMD = 0.84 pg/mL) were elevated (p < 0.05) in the melatonin-injected group. Melatonin implantation ameliorated testicular blood flow, as indicated by a significant reduction (p < 0.05) in the resistive index (RMD = 0.11) and pulsatility index (RMD = –0.15).
Conclusion
Melatonin administration can increase the reproductive performance of small male ruminants.
Keywords: goat, implantation, melatonin, meta-analysis, reproduction, sheep
Introduction
Reproductive inefficiency is a major threat to the sustainability of small ruminants, causing significant economic losses [1]. The reproductive efficacy of male animals, especially small ruminants, is equally important as that of female animals [2]. The quality of sperm accounts for reproductive success [3]. It contributes to 50% of the flock’s performance [4]. Effective management of small male ruminants before and during the breeding season is essential to reduce suboptimal reproductive performance and increase the profitability and sustainability of sheep production [5, 6]. However, only 70%–75% of rams exhibit peak reproductive performance at the beginning of the breeding season [7]. Therefore, it is necessary to devise a proper strategy to enhance the reproductive traits of small male ruminants.
Melatonin, a neurohormone secreted by the pineal gland, is a critical regulator of various physiological processes, including the regulation of circadian rhythms, antioxidant defenses, and immune modulation [8]. Melatonin administration has been intensively investigated to modulate reproductive cycles in small male ruminants. Melatonin injection has beneficial effects on testosterone production, sperm, and quality of post-thawed semen in sheep and goats during both breeding and non-breeding seasons [9–30]. Melatonin administration also mitigates sperm abnormalities in heat-stressed rams [31]. However, contradictory results have also been reported. Melatonin implantation in the ram does not correlate with sperm production or concentration [32]. Similarly, sperm quality and testosterone levels do not improve in melatonin-treated rams during the breeding season [33]. Furthermore, the administration of melatonin during breeding and non-breeding seasons has no impact on sperm and post-thawed semen quality, including motility and morphology in rams [34–37]. In addition, testosterone levels do not differ between the untreated and melatonin-treated rams during light challenges [38]. These contradictory findings necessitate a comprehensive statistical assessment to determine the influence of melatonin administration on sperm quality in small ruminants.
Meta-analysis is a statistical technique used to synthesize the results of previous studies to produce a robust quantitative conclusion [39]. It provides an unbiased and objective synthesis [40, 41]. To the best of our knowledge, no meta-analysis has been conducted on the relationship between melatonin implantation and sperm quality in small ruminants. Therefore, this meta-analysis synthesized the results of previous studies on the influence of melatonin injection on reproduction traits and sperm quality in small ruminants.
Materials and Methods
Ethical approval
Ethical approval was not necessary for this study. The preferred reporting items for systematic review and meta-analyses (PRISMA) protocols were applied in this meta-analysis, as shown in Figure-1.
Figure-1.
Selection of included studies using PRIMA protocols.
Study period and location
The meta-analysis study was conducted from August to December 2023 at Faculty of Veterinary Medicine, Gadjah Mada University, Indonesia, and National Research and Innovation Agency, Indonesia.
Search strategy
Comprehensive studies that assessed the impact of melatonin implantation on reproductive traits in small male ruminants were identified using the Science Direct, Wiley Online Library, PubMed, and Scopus databases. The search used the following keywords: “Melatonin”, “Sperm”, “Semen”, “Ram”, “Sheep”, “Buck”, and “Goat” which are connected through search queries such as “AND” and “OR”, respectively.
Inclusion and exclusion criteria
After erasing the duplication, the identified studies were excluded if they were (1) in vitro studies, (2) review studies, (3) no full-text studies, (4) no small-ruminant studies, and (5) no melatonin implantation studies. Furthermore, the criteria for selected studies were as follows: (1) The control (non-treatment) group was available; (2) measures of variance (e.g., standard deviations [SD], standard errors [SE], or confidence intervals [CI]) were provided; and (3) the influence of melatonin injection on sperm quality, post-thawed semen quality, testicular blood flow, and plasma steroid hormones in small ruminants was studied.
Extraction
The included studies are presented in Table-1 [9–38] as follows: First author’s name, year, location, species, season, dose, and research duration. The graphical data were extracted using WebPlotDigitizer (Automeris LLC, CA, USA) [42]. SD was calculated using the following formula: (1) SD = SE√N, where N is the repetition number; and (2) SD = √N × (upper CI–lower CI)/3.92, where 3.92 is the SE of the 95% CI and replaced with the t-distribution value if the sample was <60 in publications with no report of SD [43].
Table-1.
The included studies.
| No. | Author | Year | Location | Species | Season | Dose, mg | Duration, d |
|---|---|---|---|---|---|---|---|
| 1 | Abbas et al. [9] | 2021 | Pakistan | Goat | Non-breeding | 18 | 70 |
| 2 | Casao et al. [10] | 2010 | Spain | Sheep | Non-breeding | 54 | 120 |
| 3 | Casao et al. [11] | 2013 | Spain | Sheep | Non-breeding | 54 | 147 |
| 4 | Delgadillo et al. [12] | 2001 | Mexico | Goat | Non-breeding | 36 | 315 |
| 5 | Egerszegi et al. [13] | 2013 | Hungary | Sheep | Non-breeding | 18,36 | 30 |
| 6 | El-Shalofy et al. [14] | 2021 | Egypt | Sheep | Breeding | 18 | 42 |
| 7 | El-Shalofy et al. [15] | 2022 | Egypt | Sheep | Non-breeding | 18,36 | 56 |
| 8 | Gallego-Calvo et al. [16] | 2015 | Spain | Goat | Breeding and non-breeding | 54 | 60 |
| 9 | Hanif et al. [17] | 1991 | United Kingdom | Sheep | Non-breeding | 90,118 | 60 |
| 10 | Kleemann et al. [18] | 2021 | Australia | Sheep | Non-breeding | 54 | 270 |
| 11 | Kleemann et al. [19] | 2022 | Australia | Sheep | Non-breeding | 18,36,54 | 120 |
| 12 | Kokolis et al. [20] | 2000 | Greece | Sheep | Breeding and non-breeding | 54 | 105 |
| 13 | Leyva-Corona et al. [21] | 2023 | Mexico | Sheep | Non-breeding | 18,36 | 120 |
| 14 | Pool et al. [22] | 2020 | Australia | Sheep | Non-breeding | 54 | 161 |
| 15 | Rekik et al. [23] | 2015 | Jordan | Sheep | Non-breeding | 54 | 60 |
| 16 | Rosa et al. [24] | 2000 | United Kingdom | Sheep | Non-breeding | 18,36 | 42 |
| 17 | Roshan et al. [25] | 2023 | Iran | Sheep | Non-breeding | 54 | 60 |
| 18 | Samir et al. [26] | 2020 | Japan | Goat | Non-breeding | 36 | 56 |
| 19 | Tsantarliotou et al. [27] | 2008 | Greece | Sheep | Breeding and non-breeding | 18 | 105 |
| 20 | Vince et al. [28] | 2017 | Croatia | Goat | Non-breeding | 72 | 90 |
| 21 | Zarazaga et al. [29] | 2010 | Spain | Goat | Non-breeding | 54 | 210 |
| 22 | Shahat et al. [30] | 2022 | Canada | Sheep | Breeding | 36 | 49 |
| 23 | Shahat et al. [31] | 2022 | Canada | Sheep | Breeding | 36 | 49 |
| 24 | Rosa et al. [32] | 2012 | United Kingdom | Sheep | Non-breeding | 18,36 | 42 |
| 25 | Kaya et al. [33] | 2000 | Turkey | Sheep | Breeding and non-breeding | 18,36 | 66 |
| 26 | Buffoni et al. [34] | 2015 | Argentine | Sheep | Non-breeding | 54 | 90 |
| 27 | Faigl et al. [35] | 2009 | Hungary | Sheep | Non-breeding | 54 | 71 |
| 28 | Kaya et al. [36] | 2001 | Turkey | Sheep | Breeding and non-breeding | 18 | 71 |
| 29 | Pool et al. [37] | 2020 | Australia | Sheep | Breeding | 54 | 210 |
| 30 | Abecia et al. [38] | 2017 | Spain | Sheep | Breeding | 18 | 30 |
Statistical analysis
Meta-analysis and meta-regression were performed using the “metafor” package (Free Software Foundation, Inc., MA, USA) [44] and R software (R Foundation, Vienna, Austria) [45]. The influence of melatonin injection on reproductive traits and steroid hormones in small male ruminants was synthesized by computing the raw mean differences (RMDs) between untreated (control) and treated (melatonin injection) means, using the inverse of the variance for the random-effect model of the method of moments by DerSimonian and Laird [46]. RMD was selected to measure the findings in the original units [47].
Heterogeneity evaluation
We assessed heterogeneity among studies using Q test [48]. The significance level was set at p ≤ 0.10. I2 statistic was used to classify the observed heterogeneity, where I2 values <25% imply low, 25%–50% denote moderate, and >50% represent high [49]. We used Egger’s regression method to indicate publication bias [50]. Significance was defined as p ≤ 0.05.
subgroup analysis
Meta-regression was performed if the following criteria were fulfilled: (1) Heterogeneity was significant (p ≤ 0.10 or I2 > 50%), (2) there was no publication bias (p-value of Egger’s test >0.05), and (3) the number of comparisons was >10 [47]. A categorical covariate was the type of season (breeding and non-breeding). In addition, days post-treatment (days) and doses of melatonin (mg) were employed as continuous covariates. We applied the method of moments proposed by DerSimonian and Laird [46] to perform meta-regression. Subgroup analysis was implemented to evaluate RMD in the presence of significant results (p ≤ 0.05) within categorical or continuous covariates. The doses of melatonin were 18, 36, 54, and more than 54 mg. Furthermore, the days post-injection were sub-grouped into 1–31, 31–60, and >60 days post-injection.
Results
Study attributes
Thirty studies were performed in 15 countries, primarily Spain (16.67%), Australia (13.33%), and the UK (10.00%) (Table-1) [9–38]. The percentages of sheep and goats were 80% and 20%, respectively. The studies were conducted during the breeding (28.57%) and non-breeding (71.43%) seasons. In addition, melatonin doses ranged from 18 to 118 mg and the duration ranged from 30 to 315 days.
Quality of sperm and post-thawed semen
Melatonin treatment increased (p < 0.05) sperm concentration, normal morphology, viability, acrosome integrity, and DNA integrity (Table-2). All post-thawed sperm quality parameters, including progressive motility, total motility, acrosome, and DNA integrity, were increased (p < 0.05) in melatonin-injected small ruminants (Table-3).
Table-2.
Effect of implanted melatonin on sperm quality of small-ruminants.
| Item | N | Control means (SD) | RMD (95% CI) | p-value | Heterogeneity test | Egger’s test | |
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| p-value | I2 (%) | p-value | |||||
| Volume, mL | 67 | 0.90 (0.30) | −0.03 (−0.08; 0.03) | ns | <0.0001 | 78.14 | 0.099 |
| Concentration, ×109/mL | 65 | 3.68 (1.30) | 0.42 (0.24; 0.58) | <0.0001 | <0.0001 | 74.94 | 0.047 |
| Cell number, ×109/mL | 29 | 3.62 (1.57) | 0.33 (−0.18; 0.84) | ns | <0.0001 | 81.97 | 0.413 |
| Mass motility, score 1–5 | 41 | 3.19 (1.05) | −0.08 (−0.23; 0.06) | ns | <0.0001 | 84.64 | 0.002 |
| Live sperm, % | 44 | 71.63 (9.43) | 0.65 (−0.40; 1.71) | ns | <0.0001 | 73.53 | <0.0001 |
| Normal morphology, % | 50 | 75.03 (5.35) | 2.82 (1.22; 4.42) | <0.001 | <0.0001 | 88.44 | 0.850 |
| Viability, % | 33 | 71.60 (5.07) | 2.83 (1.68; 4.00) | <0.0001 | <0.001 | 50.22 | 0.070 |
| Acrosome integrity, % | 21 | 82.47 (1.79) | 4.26 (2.14; 6.37) | <0.0001 | <0.0001 | 96.65 | 0.100 |
| DNA integrity, % | 25 | 92.87 (4.26) | 1.09 (0.70; 1.49) | <0.0001 | 0.474 | 0.00 | 0.032 |
N=Number of comparisons, SD=Standard deviation, CI=Confidence of interval, I2=Inconsistency index, ns=Non-significant, RMD=Raw mean difference
Table-3.
Post-thawed semen quality between melatonin-treated and control small-ruminants.
| Item | N | Control means (SD) | RMD (95% CI) | p-value | Heterogeneity test | Egger’s test | |
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| p-value | I2 (%) | p-value | |||||
| Post-thawed total motility, % | 16 | 50.91 (11.49) | 5.62 (2.28; 8.95) | <0.001 | <0.0001 | 76.65 | 0.041 |
| Post-thawed progressive motility, % | 15 | 32.34 (10.56) | 7.90 (3.08; 12.72) | 0.001 | <0.0001 | 90.73 | 0.810 |
| Post-thawed acrosome integrity, % | 9 | 66.13 (2.94) | 8.68 (4.23; 13.12) | <0.001 | <0.0001 | 95.98 | 0.782 |
| Post-thawed DNA integrity, % | 11 | 68.35 (1.08) | 2.01 (0.65; 3.37) | 0.004 | <0.0001 | 97.04 | 0.207 |
N=Number of comparisons, SD=Standard deviation, CI=Confidence of interval, I2 =Inconsistency index,
ns=Non-significant, RMD=Raw mean difference
Sperm kinetic parameters
Total motility in melatonin-treated small ruminants increased (p < 0.05). Similarly, melatonin treatment enhanced average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and progressive motility (Table-4). However, melatonin did not influence the amplitude of lateral head displacement (p < 0.05).
Table-4.
Comparison results of sperm kinetic parameters in melatonin-treated and control small-ruminants.
| Item | N | Control means (SD) | RMD (95% CI) | p-value | Heterogeneity test | Egger’s test | |
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| p-value | I2 (%) | p-value | |||||
| Total motility, % | 27 | 74.40 (5.40) | 5.78 (2.32; 9.23) | 0.001 | <0.0001 | 98.41 | 0.789 |
| Progressive motility, % | 57 | 52.86 (4.75) | 5.28 (3.85; 6.70) | <0.0001 | <0.0001 | 99.72 | 0.007 |
| VCL, µm/s | 33 | 87.89 (9.07) | 4.09 (2.39; 5.79) | <0.0001 | <0.0001 | 85.45 | 0.080 |
| VSL, µm/s | 33 | 63.27 (7.34) | 5.61 (2.42; 8.91) | 0.001 | <0.0001 | 88.69 | 0.823 |
| VAP, µm/s | 33 | 61.80 (7.66) | 4.94 (2.08; 7.81) | 0.001 | <0.0001 | 87.67 | 0.461 |
| ALH, µm | 23 | 2.42 (0.22) | 0.02 (−0.04; 0.07) | ns | 0.039 | 37.16 | 0.962 |
N=Number of comparisons, SD=Standard deviation, CI=Confidence of interval, I2=Inconsistency index, ns=Non-significant, VCL=Curvilinear velocity, VSL=Straight-line velocity, VAP=Average path velocity, ALH=Amplitude of lateral head displacement, RMD=Raw mean difference
Reproductive steroid hormones
Testosterone levels were increased (p < 0.05) in small melatonin-implanted ruminants (Table-5). In addition, the administration of melatonin improved (p < 0.05) estradiol 17-ß levels.
Table-5.
Effect of exogenous melatonin on reproductive steroid hormones in male small-ruminants.
| Item | N | Control means (SD) | RMD (95% CI) | p-value | Heterogeneity test | Egger’s test | |
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| p-value | I2 (%) | p-value | |||||
| Testosterone, ng/mL | 209 | 5.68 (2.62) | 1.02 (0.70; 1.35) | <0.0001 | <0.0001 | 92.88 | <0.0001 |
| Estradiol 17-ß, pg/mL | 40 | 66.18 (13.55) | 0.84 (0.13; 1.54) | 0.020 | <0.0001 | 54.87 | <0.0001 |
N=Number of comparisons, SD=Standard deviation, CI=Confidence of interval, I2=Inconsistency index, ns=Non-significant, RMD=Raw mean difference
Testicular blood flow
Melatonin injection affected the resistive index (RI), peak systolic velocity (PSV), and pulsatility index (PI) (p < 0.05) (Table-6). End-diastolic velocity was not influenced by melatonin administration (p > 0.05).
Table-6.
Effect of injected melatonin on testicular blood flow parameters (pulsed-wave Doppler indices) of small-ruminants.
| Item | N | Control means (SD) | RMD (95% CI) | p-value | Heterogeneity test | Egger’s test | |
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| p-value | I2 (%) | p-value | |||||
| PSV, cm/s | 34 | 26.23 (5.94) | −2.63 (−4.24; −1.02) | 0.001 | <0.0001 | 71.08 | 0.232 |
| EDV, cm/s | 34 | 12.09 (4.06) | −0.39 (−1.18; 0.39) | ns | 0.043 | 31.53 | 0.119 |
| RI | 42 | 0.55 (1.85) | −0.11 (−0.13; −0.09) | <0.0001 | 0.017 | 34.36 | 0.499 |
| PI | 42 | 0.79 (2.19) | −0.15 (−0.18; −0.10) | <0.0001 | 0.002 | 42.62 | 0.750 |
N=Number of comparisons, SD=Standard deviation, CI=Confidence of interval, I2=Inconsistency index, ns=Non-significant, PSL=Peak systolic velocity, EDV=End-diastolic velocity, RI=Resistive index, PI=Pulsatility index, RMD=Raw mean difference
Publication bias and meta-regression analysis
Heterogeneity was significant (p < 0.05) for all parameters of sperm quality, post-thawed semen quality, sperm motility, reproductive steroid hormones, and testicular blood flow parameters (Tables-2-6). On the other hand, publication bias was not found (p > 0.05) on sperm normal morphology, sperm total motility, sperm viability, acrosome integrity, VCL, VSL, VAP, PSV, RI, PI, post-thawed progressive motility, post-thawed acrosome integrity, and post-thawed DNA integrity. Meta-regression was performed when the effect size and heterogeneity were significant and publication bias was not present.
The melatonin dose explained 65.17%, 100%, 93.79%, and 47.71% of the observed heterogeneity for acrosome integrity, RI, PI, and VCL, respectively (p < 0.0001) (Table-7). Days post-melatonin treatment explained 58.00% (p < 0.001), 26.96% (p = 0.034), and 44.64% (p = 0.006) of the observed heterogeneity for sperm viability, RI, and PI, respectively. Moreover, melatonin dose explained 31.71% (p = 0.037), 36.53% (p = 0.001), an d 5.44% (p = 0.002) of the observed heterogeneity for VAP, post-thawed progressive motility, and post-thawed DNA integrity.
Table-7.
Meta-regression comparing the association between covariates and measured outcomes.
| Parameter | Covariates | QM | p-value | R2 (%) |
|---|---|---|---|---|
| Sperm normal morphology | Dose | 0.01 | ns | 1.32 |
| Days post-injection | 2.57 | ns | 6.39 | |
| Season | 21.03 | <0.0001 | 24.97 | |
| Sperm viability | Dose | 1.10 | ns | 8.44 |
| Days post-injection | 14.20 | <0.001 | 58.00 | |
| Season | NA | NA | NA | |
| Sperm total motility | Dose | 0.16 | ns | 0 |
| Days post-injection | 0.27 | ns | 16.73 | |
| Season | 4.26 | 0.040 | 45.54 | |
| Acrosome integrity | Dose | 34.59 | <0.0001 | 65.17 |
| Days post-injection | 0.20 | ns | 0 | |
| Season | 6.58 | 0.010 | 0 | |
| PSV | Dose | 0 | ns | 0 |
| Days post-injection | 0.10 | ns | 0 | |
| Season | 0.39 | ns | 0 | |
| RI | Dose | 27.53 | <0.0001 | 100 |
| Days post-injection | 4.47 | 0.034 | 26.96 | |
| Season | 2.19 | ns | 12.14 | |
| PI | Dose | 28.09 | <0.0001 | 93.79 |
| Days post-injection | 7.59 | 0.006 | 44.64 | |
| Season | 35.80 | <0.0001 | 100 | |
| VCL | Dose | 31.62 | <0.0001 | 47.71 |
| Days post-injection | 3.15 | ns | 0 | |
| Season | 41.28 | <0.0001 | 49.37 | |
| VSL | Dose | 1.46 | ns | 17.02 |
| Days post-injection | 0.002 | ns | 0 | |
| Season | 16.79 | <0.0001 | 32.35 | |
| VAP | Dose | 4.34 | 0.037 | 31.74 |
| Days post-injection | 0.24 | ns | 0 | |
| Season | 18.25 | <0.0001 | 45.94 | |
| Post-thawed progressive motility | Dose | 11.42 | <0.001 | 36.53 |
| Days post-injection | 0.05 | ns | 0 | |
| Season | 10.60 | 0.001 | 35.90 | |
| Post-thawed DNA integrity | Dose | 4.76 | 0.002 | 5.44 |
| Days post-injection | 1.86 | ns | 36.91 | |
| Season | 4.76 | 0.002 | 5.44 |
QM=Coefficient of moderators, R2=Amount of heterogeneity accounted for by covariate, NA=Non-available, ns=Non-significant, PSV=Peak systolic velocity, RI=Resistive index, PI=Pulsatility index, VCL=Curvilinear velocity, VSL=Straight-line velocity, VAP=Average path velocity
The season of the experiment explained (p < 0.001) 24.97%, 100%, 49.37%, 32.35%, and 45.94% for normal sperm morphology, PI, VCL, VSL, and VAP, respectively. In addition, 35.39% (p = 0.001) and 5.44% (p = 0.002) of the observed heterogeneity for post-thawed progressive motility and post-thawed DNA integrity were explained by the season of the experiment (Table-7).
subgroup analysis
The dose of 36 mg melatonin increased (p < 0.0001) VCL (Figure-2a; RMD = 10.72), VAP (Figure-2b; RMD = 11.74), acrosome integrity (Figure-2c; RMD = 8.93), post-thawed progressive motility (Figure-2d; RMD = 13.50), and post-thawed DNA integrity (Figure-2e; RMD = 2.84). Similarly, 54 mg dose enhanced acrosome integrity (p = 0.04) (Figure-2c; RMD = 11.48). However, 18- and 36-mg melatonin reduced RI (RMD = –0.04; p = 0.02 and RMD = –0.14; p < 0.0001, respectively) (Figure-2f). PI decreased (p < 0.0001) at 36 mg injection of melatonin (Figure-2g; RMD = –0.18).
Figure-2.
Subgroup analysis of the effect of dose melatonin injection on (a) straight-line velocity, μm/s, (b) average path velocity, μm/s, (c) acrosome integrity, %, (d) post-thawed progressive motility, %, (e) post-thawed DNA integrity, %, (f) resistive index, and (g) pulsatility index.
RI was reduced (p < 0.0001) on days 1–30 post-melatonin treatment (RMD = –0.12) and 31–60 post-melatonin treatment (RMD = –0.13) (Figure-3a). Similarly, PI decreased (p < 0.0001) on 1–30 days (RMD = –0.16) and 31–60 days post-melatonin treatment (RMD = –0.18) (Figure-3b). Sperm viability was enhanced on days 1–30 (RMD = 1.73; p = 0.005) and 31–60 (RMD = 5.51; p < 0.0001) post-melatonin treatment (Figure 3c).
Figure-3.
Subgroup analysis of the season type of effect of melatonin administration on (a) resistive index, (b) pulsatility index, and (c) sperm viability, %.
Melatonin injection during the breeding season enhanced (p < 0.0001) normal sperm morphology (RMD = 9.70), sperm total motility (RMD = 8.57), acrosome integrity (RMD = 7.83), VCL (RMD = 9.73), VSL (RMD = 13.90), VAP (RMD = 11.34), post-thawed progressive motility (RMD = 12.46), and post-thawed DNA integrity (RMD = 2.84) (Table-8). Moreover, melatonin implantation decreased PI (p < 0.0001) in the breeding (RMD = –0.09) and non-breeding (RMD = –0.27) seasons.
Table-8.
Subgroup analysis of season type of the effect of melatonin injection on sperm quality, sperm motility, and testicular blood flow parameters.
| Parameter | Breeding | Non-breeding | ||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| N | RMD (95% CI) | p-value | N | RMD (95% CI) | p-value | |
| Sperm normal morphology | 9 | 9.70 (6.44; 12.96) | <0.0001 | 41 | 1.22 (−00.36; 2.80) | ns |
| Sperm total motility | 11 | 8.57 (4.80; 12.34) | <0.0001 | 16 | 3.02 (−00.65; 6.70) | ns |
| PI | 15 | −0.09 (−0.13; −0.07) | <0.0001 | 27 | −00.27 (−032; −00.22) | <0.0001 |
| Acrosome integrity | 9 | 7.83 (4.22; 11.43) | <0.0001 | 12 | 1.54 (−01.63; 4.71) | ns |
| VCL | 9 | 9.73 (7.44; 12.02) | <0.0001 | 24 | 0.48 (−01.18; 2.13) | ns |
| VSL | 9 | 13.90 (9.09; 18.71) | <0.0001 | 24 | 1.62 (−01.74; 4.99) | ns |
| VAP | 9 | 11.34 (7.62; 15.07) | <0.0001 | 24 | 1.08 (−01.79; 3.96) | ns |
| Post-thawed progressive motility | 9 | 12.46 (7.63; 17.30) | <0.0001 | 6 | −01.67 (−08.67; 5.33) | ns |
| Post-thawed DNA integrity | 8 | 2.84 (1.32; 4.37) | <0.001 | 3 | −00.62 (−03.34; 2.09) | ns |
N=Number of comparisons, RMD=Raw mean difference, CI=Confidence of interval, ns=Non-significant, PI=Pulsatility index, VCL=Curvilinear velocity, VSL=Straight-line velocity, VAP=Average path velocity
Discussion
Melatonin modulates the release of gonadotropin-releasing hormone [51], a key regulator of reproductive physiology. It is actively transported into the testes [52], modulating various cellular processes involved in spermatogenesis and steroidogenesis [53, 54]. Melatonin administration enhances spermatogenesis [55].
Our meta-analysis revealed that treatment with melatonin enhances sperm quality in small ruminants, including viability, concentration, normal morphology, acrosome, and DNA integrity. These findings are similar to those reported by Abbas et al. [9], who reported that injection of melatonin increases the concentration, normal morphology, acrosome, and DNA integrity of sperm in rams. Moreover, motility, acrosome, and DNA integrity in the post-thawed semen of melatonin-treated small ruminants were also increased. Shahat et al. [30] discovered that injection of melatonin improves post-thawed motility, acrosome, and DNA integrity of rams.
Blood circulation is crucial, especially for testicular function [56]. Enhanced testicular blood flow increases the supply of oxygen and nutrients to the testes [57]. Melatonin enhances testicular blood flow [26] and is correlated with heightened sperm quality in rams [31]. Resistance (RI) and perfusion (PI) indices are key indicators extensively used for evaluating testicular blood flow in various animals [58–61]. Reduced RI and PI values indicated elevated testicular blood flow [62]. This study also found that elevated sperm quality is linked to low RI and PI values. Therefore, the increase in sperm and post-thawed semen quality in melatonin-treated small ruminants can be attributed to the enhancement of testicular blood supply.
This study showed that melatonin implantation improves sperm motility and velocity of small ruminants. Egerszegi et al. [13] and Casao et al. [10] also found that injection of melatonin increases sperm motility and ram velocity in the out-of-season period. Moreover, Shahat et al. [31] found that sperm motility and velocity of melatonin-treated rams subjected to mild heat-stressed challenges increased during breeding seasons.
RI and PI are negatively correlated with the progressive motility of sperm [62]. Decreased arterial blood flow to the testes impairs mitochondrial energy processes, preventing spermatogenesis. This dysfunction of the energetic pathway reduces sperm motility [63]. Our results revealed that sperm kinetic parameters increased concomitantly with the enhancement of testicular blood flow. Thus, we suggest that melatonin modulates testicular blood flow, resulting in increased mitochondrial energy production and enhanced sperm motility.
In this study, we found that testosterone and estradiol-17β levels are elevated in melatonin-treated small ruminants. These findings are consistent with those of previous studies where testosterone levels were elevated in melatonin-treated rams and bucks [16, 31]. Melatonin affects testosterone production through the anterior pituitary gland and ameliorates Leydig cell function to promote testosterone secretion in sheep [64]. Samir et al. [26] found that melatonin elevates testosterone production in Shiba bucks through the hypothalamus-pituitary axis. Moreover, Casao et al. [10] discovered that the enhancement of antioxidant enzymes induces the elevation of testosterone and estradiol-17β levels in melatonin-treated rams.
Furthermore, our study demonstrated that melatonin implantation increases PSV and decreases RI and PI, indicating enhanced testicular blood supply. RI and PI levels are regulated by plasma estradiol-17β [65]. Bollwein et al. [66] found that estradiol-17β levels, but not testosterone levels, control testicular blood flow in stallions. Moreover, Salama et al. [67] discovered that enhanced testicular blood flow in melatonin-treated canines is triggered by elevation of estradiol-17β levels. Similarly, El-Shafoly et al. [15] reported that melatonin-injected rams show a reduction in RI and PI levels accompanied by an increase in estradiol-17β levels. Interestingly, our study also demonstrated a correlation between decreased RI and PI and enhancement of estradiol-17β levels. Therefore, we suggest that melatonin administration improves testicular blood flow in small ruminants by modulating estradiol-17β levels.
Conclusion
These results indicate that melatonin administration can improve sperm quality in small ruminants. The best outcomes for acrosome integrity, VCL, post-thawed progressive motility, and post-thawed DNA integrity were achieved with 36 mg melatonin injection during the breeding season. As evidenced by a reduction in RI and PI, optimal results for testicular blood supply were attained with 36 mg melatonin implantation during the breeding season within 1–60 days post-injection. Moreover, melatonin administration was associated with superior results in normal morphology, motility, VSL, and VAP during the breeding season.
Authors’ Contributions
AB: Designed the study and wrote and revised the manuscript. SH: Screened studies, analyzed data, and edited the manuscript. RW: Scrutinized included studies and edited and reviewed manuscript. HK, WW, BH, IMM, AI, and DDL: Extracted data and reviewed the manuscript. All authors have read, reviewed, and approved the final manuscript.
Acknowledgments
We would like to thank the Faculty of Veterinary Medicine, Gadjah Mada University, and the National Research and Innovation Agency for supporting this collaboration study. The study did not receive any funds.
Competing Interests
The authors declare that they have no competing interests.
Publisher’s Note
Veterinary World remains neutral with regard to jurisdictional claims in published institutional affiliation.
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