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males and 10000 females. In each generation, 10 bulls and 50 cows were chosen as parents of the male progeny, and 20 bulls and 10000 cows were chosen as parents
of the female progeny (with no selection on the dam to dam path). Mating was random resulting in random variation of progeny size among parents, i.e. the sire and dam
of a given newborn were randomly chosen in the corresponding lists of parents. Simulation of genetic values and EBV We considered two traits A and B. Trait A corresponded to
a production trait which had been recently and intensively selected and improved (such as milk production in dairy cattle). Trait B corresponded to a functional trait which had deteriorated because
of a negative correlation with trait A (e.g. fertility or longevity). The genetic standard deviation of each trait (σA and σB, respectively) was set to 1 and the correlation between
traits (ρ) was set to -0.3. For each trait, an additive polygenic model was assumed and the simulation of correlated genetic values was based on the bivariate normal distribution (see,
e.g. ). At generation 0 (base population), genetic values for trait A were randomly and independently drawn from a N (0, 1) distribution. For a given individual (i), the genetic
value for trait B (Bi) was generated from its value for trait A (Ai): where βi is a N (0, 1) random number independent of Ai. In the following generations,
genetic values of individual i were simulated from the genetic values of its sire (Ap and Bp) and its dam (Am and Bm), taking into account the parent's coefficients of
inbreeding (Fp and Fm, resp.) [12,13]: In these equations, γi and δi are two numbers randomly drawn from a N (0, 1) bivariate normal distribution with a correlation equal to
ρ. EBV were directly simulated from genetic values, assuming an evaluation procedure leading to an accuracy (CD = square of the correlation between the EBV and the true genetic value)
equal to 0.6 for bulls and 0.4 for cows, whatever the trait and the generation considered. Therefore, the EBV of a given individual for trait A (EBVAi) and for trait
B (EBVBi) were computed as follows: where εi and ϕi are two independent numbers drawn from a N(0, 1) distribution. Finally, a Total Merit Index (TMIi) was computed, weighting the
two EBV by wA and wB = 1 - wA, respectively: Sampling and use of cryopreserved semen Simulations comprised two stages. During stage 1 (generations 0 to 8), the lists
of parents were selected based on their EBV for trait A only, without considering the evolution of the genetic mean for trait B or the average coefficient of inbreeding. During
stage 2 (generations 9 to 12), the bulls were also used to improve trait B or to introduce genetic diversity in the breed. During stage 1, the semen of some
bulls was sampled and cryopreserved if the animals fulfilled one of the three following conditions, which correspond to the current sampling rules of the French National Cryobank for type "II"
(original bulls) : - (i) EBVA is three standard deviations above or below the mean of the generation, - (ii) EBVB is two standard deviations above the mean of the
generation (trait B is considered as a functional trait and for functional traits, only animals above the average are considered), - (iii) the bull is a sire of sires with
no male offspring selected after the evaluation process (these bulls were actually selected with one generation lag). To check the validity of this elaborate sampling method, we tested a simpler
sampling method (similar to the one used in the Netherlands), where the semen of all young bulls is stored in the cryobank. In the simulations performed here, we investigated the
impact of a one-time use (i.e. during a single generation) of cryopreserved semen. At generation 9, four bulls with cryopreserved semen were selected (hereafter referred to as 'cryobank bulls'), these
bulls fulfilling one of the following conditions either (i) they are the best cryobank bulls for the TMIi or (ii) they have the lowest average kinship with the existing population
(males and females taken together). We studied the impact of various selection orientations (use of cryopreserved semen, conservation of male lines, etc.) only on the male path, because applying the
above conditions on the female path would be much more restrictive, less effective, and would require a larger amount of semen, all the more since the number of doses is
generally limited in cryobanks (200, in France) . For these reasons, we considered that cryobank bulls were used only to procreate young bulls for progeny testing. The 9th generation of
young bulls was then generated using either the bulls from the cryobank or the group of 10 sires selected as described in previous sections. Depending on the scenario (see following
section), 0, 40 or 80 individuals (among the 100 newborn bull calves) were sired randomly by one of the four selected cryobank bulls. Simulation scenarios and results Six simulation scenarios
were completed with two main options (Table 1). Table 1. Description of simulation scenarios Firstly, in scenario "b", emphasis was put on the selection of both traits B and A.
To achieve this goal, three methods were compared: - b1: at generation 9, the four bulls with the highest TMI (wB = 0.5) were used to sire 40% of the
young bulls, while the selection criterion during stage 2 remained unchanged (improving EBVA). The other young bulls were sired by bulls randomly sampled within the group of 10 sires; -
b2: at generation 9, no cryobank bull was used, and during stage 2, TMI (wB = 0.5) was used as the selection criterion instead of EBVA; - b3: at generation
9, the four cryobank bulls with the highest TMI were used to sire 40% of the young bulls, and during stage 2, TMI was used as the selection criterion instead
of EBVA. To test more or less drastic selection changes, scenario b3 was tested with an increasing weight given to trait B (wB increasing from 0.5 to 1). Secondly, in
scenarios "d", emphasis was put on genetic variability while trait A remained the breeding goal. Three methods were also compared: - d1: at generation 9, the four cryobank bulls having
the lowest kinship with the existing population (scenario b1) were used to sire 40% of the young bulls; - d2: at generation 9, no cryobank bull was used, while during
stage 2, the progenies on the sire to sire path were given the same size i.e. for each sire of sires, 10 male offspring were created among which those with
the two best EBVA became the sires of dams and that with the best EBVA became a sire of sires; - d3: at generation 9, the four cryobank bulls having
the lowest kinship with the existing population (scenario b1) were used to sire 40% of the young bulls, while during stage 2, selection was used to equalise progeny sizes on
the sire to sire path. Simulations were performed with 1000 runs for each scenario. For each generation, individual inbreeding coefficients and genetic values were computed and averaged for the entire
male and female populations. The individual coefficients of kinship were also computed and averaged over males only and over the entire populations. The proportion of genes originating from cryobank bulls
was computed on the basis of the gene dropping procedure (one locus averaged over the 1000 runs). Stage 1: evolution of selected traits, diversity loss, and sampling of cryobank bulls
As expected, the results of the different scenarios did not differ significantly for generations 0 to 8 given that in stage 1, the conditions were the same whatever the option
chosen, (here we present results averaged over the 1000 runs of one scenario only). With the parameters chosen for the simulation, each sire of sires had on average 10 male
offspring (across sires standard deviation s.d. = 2.9) and each sire of dams had on average 500 female offspring (across sires s.d. = 21.6). As expected (see Figure 1), selection
on trait A during stage 1 led to a major increase in the mean of this trait (+ 6.7 initial genetic standard deviation) from generation 0 to 8, while at
the same time, the mean of B decreased to a lesser extent (-2 initial genetic standard deviation). The average coefficient of inbreeding increased simultaneously. Young bulls were slightly more inbred
than cows, as they originated from a smaller number of sires and dams. In parallel (generation 0 to 8), the average coefficient of kinship among the young bulls and among
the entire population increased to 8.1% and 6.9%, respectively. Figure 1. Changes in genetic values (a) and in genetic diversity (b) (scenario b1). Dotted lines: young bulls; solid lines: whole
population; red: trait A, blue: trait B; green: average between A and B; purple: inbreeding F; pink: kinship Φ. An average of 31 cryobank bulls was sampled per replicate, 58%
being sampled because of outstanding EBVB (see Table 2). Table 2 shows that cryobank bulls chosen for their genetic diversity were generally born earlier than others, which can be explained
by the fact that they were chosen with one generation lag compared to other sampling procedures. Table 2. Average number and birth generation of bulls selected for conservation Stage 2
in scenarios b: change in breeding goals As shown in Figure 1, introducing cryobank bulls with exceptional TMI without changing the selection criterion during stage 2 (scenario b1) had a
temporary impact on traits A and B as well as on the diversity indicators of the young bulls. At the whole population level, the impact was negligible, since young bulls
sired by cryobank bulls were rarely subsequently selected as sires: three generations after introduction (generation 12), the cryobank contribution to genetic diversity was less than 3% (Table 3). Table 3.
Origin and impact of cryobank bulls used in the different scenarios When TMI was used as a selection criterion (considering wB = 0.5), without using cryobank bulls (scenario b2), there
was a per generation increase in the mean of trait B from generation 9 on (b1: -0.3 vs b2: +0.4), while the genetic gain for trait A decreased (b1: +1.0
vs b2: +0.4, see additional file 1). The change in breeding goals had no impact on diversity indicators. Additional file 1. Changes in genetic values (a) and in genetic diversity
(b) (scenario b2). The data represent the simulation results for scenario b2. Dotted lines: young bulls; solid lines: whole population; red: trait A; blue: trait B; green: average between A
and B; purple: inbreeding F; pink: kinship Φ. Format: PDF Size: 58KB Download file This file can be viewed with: Adobe Acrobat Reader Combining the use of cryobank bulls and
TMI as a selection criterion (scenario b3 for wB = 0.5) resulted in a slight but significant (P < 0.001) reduction in average kinship (-0.3% between scenario b2 and b3,
with 40% of the males from generation 9 sired by cryobank bulls, see additional file 2). Concerning the selected traits, the genetic gain for trait A decreased slightly when cryobank
bulls were used (-0.12 between scenarios b2 and b3, P < 0.001), while the genetic gain for trait B increased slightly (+0.06 between scenarios b2 and b3, P = 0.02).
These tendencies increased slightly when 80% of the males from generation 9 were sired by cryobank bulls (see additional file 2). According to the results from Table 3, cryobank bulls
contributed to 6.5% of the diversity three generations after their introduction. It should be noted that the cryobank bulls used were generally sampled in recent generations, their average birth generation
being 6.6 (Table 3). Additional file 2. Changes in genetic values (a) and in average kinship (b) when trait B was added to selection goals. The data represent the simulation
results when selection is redirected with a new trait accounting for 50% of the total merit index and when the use of semen from cryobank bulls is increased. Scenario b3
and whole population are considered with the weight wB given to trait B accounting for 50% of the total merit index and an increased use of the semen from cryobank
bulls. Brown: no cryobank bull is used (scenario b2); red: cryobank bulls are used to produce 40% of sons (scenario b3); yellow: cryobank bulls are used to produce 80% of
sons; o: genetic value for trait A; ♦: genetic value for trait B; dotted line: average genetic value between A and B; x: kinship Φ. Format: PDF Size: 57KB Download
file This file can be viewed with: Adobe Acrobat Reader As a result of the increased weight of trait B within TMI (see Figure 2), there was a per generation
increase in genetic gain for trait B, while there was a slightly lower increase or even a decrease in genetic gain for trait A, as well as in average kinship,
when trait B accounted for more than 80% of EBV. When only trait B was taken into account for TMI, the genetic value of traits A and B reached 4.7
and 1.37, respectively at generation 12 (versus 8.4 and -0.41 respectively when wB = 0.5), while average kinship reached 8.9% at generation 12 (versus 11.9% when wB = 0.5). Figure
2. Changes in genetic values (a) and in average kinship (b), when trait B was added to selection goals. Scenario b3 and whole population are considered with the weight wB
of trait B increasing for computation of the total merit index. Black: wB = 0 (scenario b1); brown: wB = 0.5; red: wB = 0.6; orange: wB = 0.7; green:
wB = 0.8; light blue: wB = 0.9; dark blue: wB = 1; o: genetic value for trait A; ♦: genetic value for trait B; x: kinship Φ. Stage 2
in scenarios d: improvement in genetic diversity As shown in Figure 3, the use of cryobank bulls with a minimised kinship with the current generation (scenario d1), had no impact
if the selection policy was not modified, since none of the offspring of the cryobank bulls were selected as sires. Equalising progeny sizes on the sire to sire path alone
(scenario d2) decreased diversity a little less (in generation 12, Φ = 12% for scenario d1 and Φ = 11% for scenario d2), with an almost negligible impact on genetic
progress. Combining this option with the introgression of cryobank bulls (scenario d3) resulted in a significant reduction in average kinship (-2% in comparison to d1). Under such a scenario, the
genetic mean of trait B also increased slightly (+0.3 between scenario d1 and d3, P < 0.001), while that of trait A and the average of both traits decreased slightly
(-0.08 and -0.02 respectively, between scenarios d1 and d3, P < 0.001). It should be noted that most of the cryobank bulls used originated from the founder population, their average
birth generation being 0.3 (Table 3). Figure 3. Changes in genetic values (a) and in average kinship (b), when the aim was to manage genetic diversity. The whole population is
considered Brown: no change in selection; cryobank bulls used to produce 40% of male offspring (scenario d1); red: conservation of male lines (scenario d2) (curve overlapping the preceding one); yellow:
conservation of male lines and cryobank bulls used to produce 40% of male offspring (scenario d3); o: genetic value for trait A; ♦: genetic value for trait B; dotted line:
average genetic value between A and B; x: kinship Φ. Modifying which bulls entered the cryobank by preserving semen for all the young bulls did not significantly alter the results
of scenarios b3 and d3, either for the selected traits or for kinship evolution (data not shown). It should be noted that in this case, the average birth generation of
the cryobank bulls used was 7, in scenario b3 (instead of 6.6, in the first cryobank sampling method), and 0, in scenario d3 (instead of 0.3, in the first cryobank
sampling method). In this study, we assessed the impacts of using cryopreserved bull semen either to redirect selection or to improve the genetic variability of a selected cattle breed. Simulation
parameters were chosen as a compromise between realism in the scenarios, their applicability, and the simplicity of the model. For instance, with respect to the choice of population size, a
breed with 20 breeding males and 10000 potential dams could be considered quite small, especially with reference to the FAO endangerment status . In our simulation, sires and dams were
randomly chosen from lists of reproducers. This differs significantly from what occurs in real breeds, in which an unbalanced use of reproducers is frequently the case, leading to a reduced
size of the effective population. In terms of effective size, our breed would correspond to a much larger population with a similar inbreeding rate per generation (1.07%) to that found
in real dairy cattle breeds e.g. . Concerning sampling conditions in the simulations, as mentioned above, the procedure chosen to select bulls for cryopreservation is similar to that currently applied
in France. This choice was made to test if bulls selected this way could be effectively used in a selected breed. Compared to the case in which all young bulls
are sampled for cryopreservation (which corresponds more or less to the current procedure in the Netherlands), the results were basically the same. This shows that the French sampling procedure is
reasonably efficient to select useful bulls, and could be applied in situations when only a limited number of semen samples can be stored in a cryobank (for financial reasons, for
instance). One of the main conclusions of this study is that using cryopreserved semen is relevant for a breed for which major changes in selection objectives or practices are considered.
Since genetic progress is rapid in dairy cattle breeds (e.g. ), a bull for which semen has been stored for a few generations, is likely to have a lower genetic